KR20240052771A - Lymphocyte potency assay - Google Patents

Abstract

The present disclosure provides methods and materials useful for measuring the antitumor potency of lymphocytes, such as tumor infiltrating lymphocytes.

Description

Lymphocyte potency assay

Related applications

This application is filed under U.S. Provisional Application No. 63/241,768 (filing date: September 8, 2021), U.S. Provisional Application No. 63/291,655 (filing date: December 20, 2021), and U.S. Provisional Application No. 63/391,118 (filing date: 2021) 35 U.S.C. (July 21, 2011). § 119(e), each of which is incorporated herein in its entirety.

Reference to Electronic Sequence Listing

The contents of the electronic sequence listing (K071370018WO00-SEQ-HJD.xml; size: 21,568 bytes; creation date: September 7, 2022) are incorporated herein by reference in their entirety.

Technology field

The present disclosure relates to methods and compositions useful for assessing the antitumor potency of lymphocytes, such as tumor infiltrating lymphocytes.

Lymphocytes are white blood cells essential to the immune system. Tumor-infiltrating lymphocytes (TILs) are white blood cells, including T cells and B cells, that leave the bloodstream and migrate toward the tumor. The presence of lymphocytes in tumors is often associated with better clinical outcome, and indeed lymphocytes, such as TILs, have been implicated in the killing of tumor cells. Lymphocytes are routinely used in adoptive cell therapy (ACT) to treat certain types of cancer. For example, adoptive transfer of TILs is a powerful approach for the treatment of bulky and refractory cancers, especially in patients with poor prognosis. In ACT, cells are expanded ex vivo and their potency must be characterized before being reinjected into the patient.

In particular, TIL potency assays that measure direct or indirect biological activity associated with TILs are particularly important for ACT using TILs, given the wide heterogeneity of TIL/tumor specificity between patients. Additionally, the U.S. Food and Drug Administration (FDA) requires TIL potency testing to ensure the quality of individual TIL products for use in ACT.

Importantly, not all TIL potency assays are considered biologically relevant by virtue of the assay's biological context or assay endpoints. With increasing regulatory guidance, TIL potency assay readouts must be correlated with in vivo function (e.g., tumor recognition and cell killing) to translate into clinical potency. Assays can use non-cell-based TIL stimulation approaches and can be based on T cell properties instead of cytotoxicity (e.g., using an interferon gamma (IFN-γ) release assay to assess TIL potency), but the In vitro TIL potency assays based on tumor cell-mediated activation are considered to accurately represent TIL potency and have the clearest correlation with clinical efficacy in the field when surrogate endpoints including cytotoxicity or IFN-γ release are used ( See, for example, de Wolf et al. Cytotherapy 2018 May;20(5):601-622. Such biologically relevant TIL potency assays are currently limited in the field.

The present disclosure provides lymphocyte potency assays, such as the TIL potency assay (also referred to herein as the TIL antitumor potency assay), that can be used to assess the ability of lymphocytes to generate clinically relevant anti-tumor responses. In some embodiments, the present disclosure provides immortalized cells (e.g., tumor cells) comprising molecules that activate lymphocytes, such as molecules that activate T cells (e.g., molecules that bind T cell antigens). do. This interaction between lymphocytes and immortalized cells, such as tumor cells, can be used, for example, to evaluate the potency of lymphocytes as anti-tumor therapy for cancer. Prior to the assays described herein, most lymphocyte (e.g., TIL) potency assays were based on non-cell-based lymphocyte activation and cytokine (e.g., IFN-γ) release assays as measures of lymphocyte activity and only It can be considered a physiologically irrelevant stimulus for investigating cytolytic function.

The present disclosure provides data showing that tumor cells, e.g., human melanoma A375 cells, engineered to express membrane-associated anti-CD3 (OKT3) antibodies can be used to activate TIL anti-tumor function. . OKT3 is an activating antibody used in its soluble form or bound to beads to activate lymphocytes tethered to magnetic beads. Unexpectedly, the process of reducing the affinity of A375-pKSQ367 cells for TILs by incorporating a mutation in the OKT3 antibody that reduces the affinity of the membrane-associated binding domain for TILs resulted in, for example, three-dimensional in vitro tumor spheroid function. It serves to improve the usefulness of the assay for evaluating TIL efficacy.

The present disclosure also provides that, in some embodiments, two-dimensional monolayer cell cultures are more suitable for assessing the antitumor activity of lymphocytes, while in some embodiments three-dimensional multilayer spheroid cultures are suitable for evaluating different lymphocyte populations, such as edited TILs. Provides data indicating that TILs are more suitable for assessing functional properties of versus unedited TILs. The spheroid setup described herein allows the identification and assessment of several aspects of TIL function/biology, including cytotoxicity, cytokine production, proliferation, and phenotypic changes. On the targeting side, the spheroid dimension provides higher levels of cell-cell contact and interactions closer to those that occur in vivo. Without wishing to be bound by theory, the potency assays provided herein provide an elegant and complex cellular microenvironment to compare and contrast changes in morphological properties as well as other cellular properties.

Aspects of the disclosure provide a method of assessing the potency of a lymphocyte (e.g., a T cell), comprising co-culturing a lymphocyte (e.g., a T cell) and an immortalized cell, wherein the immortalized cell comprising a molecule that activates lymphocytes (e.g., T cells) and assessing the potency of the lymphocytes. In some embodiments, the lymphocytes are not engineered (e.g., do not contain non-naturally occurring genomic modifications).

Another aspect of the disclosure provides a method of assessing the potency of a TIL, comprising co-culturing a TIL and an engineered tumor cell, the engineered tumor cell comprising a molecule that activates a T cell. and evaluating the effectiveness of the TIL.

Another aspect of the disclosure provides a method of assessing the potency of polyclonal T cells, comprising co-culturing a polyclonal T cell and an immortalized cell, wherein the immortalized cell comprises a molecule that activates the T cell. , and evaluating the potency of polyclonal T cells.

In some embodiments, the molecule binds a T cell antigen. In some embodiments, the TIL expresses a T cell antigen. In some embodiments, the polyclonal T cells express a T cell antigen. In some embodiments, the T cell antigen is CD3 antigen. In some embodiments, the immortalized cell expresses the molecule.

In some embodiments, the molecule is an antibody or antibody fragment. For example, the antibody fragment can be selected from single chain variable fragment (scFv), F(ab')2 fragment, Fab fragment, Fab' fragment and Fv fragment. In some embodiments, the antibody fragment is an scFv. In some embodiments, the antibody or antibody fragment is an OKT3 antibody or OKT3 antibody fragment, respectively. In some embodiments, the OKT3 antibody fragment is membrane bound OKT3 (mOKT3) scFv. For example, mOKT3 scFv may be a low affinity mOKT3 scFv variant.

In some embodiments, the molecule binds CD3 with a KD that is lower than the dissociation constant (KD) of mOKT3 scFv, where the KD of mOKT3 scFv is about 5×10 ^-10 M. In some embodiments, the low affinity mOKT3 scFv variant comprises an amino acid sequence with R55 and Y57 mutations relative to the amino acid sequence of SEQ ID NO:2. In some embodiments, the low affinity mOKT3 scFv variant comprises an amino acid sequence with the R55M and Y57A mutations relative to the amino acid sequence of SEQ ID NO:2. In some embodiments, the low affinity mOKT3 scFv variant binds CD3 with a KD that is at least 250-fold lower than the dissociation constant KD of the mOKT3 scFv. In some embodiments, the low affinity mOKT3 scFv variant comprises an amino acid sequence with the R55L and Y57T mutations relative to the amino acid sequence of SEQ ID NO:2. In some embodiments, the low affinity mOKT3 scFv variant binds CD3 with a KD that is at least 1000-fold lower than the dissociation constant KD of the mOKT3 scFv.

In some embodiments, the molecule is selected from phytohemagglutinin (PHA) and concanavalin A (ConA). In some embodiments, the molecule is a bacterial superantigen, such as staphylococcal enterotoxin B (SEB). In some embodiments, the molecule is a membrane tethered molecule.

In some embodiments, the TIL is an engineered TIL (eTIL). In some embodiments, the eTIL is an edited eTIL. In some embodiments, the edited eTIL includes genomic modifications.

In some embodiments, polyclonal T cells include neoantigen-specific T cells.

In some embodiments, polyclonal T cells are derived from peripheral blood.

In some embodiments, polyclonal T cells are derived from bone marrow.

In some embodiments, the immortalized cells comprise a genetic population of immortalized cells. In some embodiments, the immortalized cells are immortalized human cells. In some embodiments, the immortalized cells are engineered (e.g., human) cancer cells. In some embodiments, the engineered cancer cells are selected from engineered melanoma cells, engineered colorectal cancer cells, engineered cholangiocarcinoma cells, and engineered breast cancer cells.

In some embodiments, the co-culture is for at least 4 hours (e.g., about 4 to 6, about 4 to 8, about 4 to 12 hours, about 4 to 18 hours, or about 4 to 24 hours). In some embodiments, the co-culture is for at least 12 hours. In some embodiments, the co-culture is for at least 24 hours, at least 48 hours, or at least 72 hours. In some embodiments, the co-culture is for about 1 day, about 1 to 3 days, about 1 to 4 days, about 1 to 5 days, about 1 to 6 days, or about 1 to 7 days.

In some embodiments, assessment of efficacy includes measuring surrogate markers of TIL reactivity to the tumor, including release of effector cytokines such as IFN-γ or expression of CD107a. In some embodiments, assessing efficacy includes measuring growth of immortalized cells. In some embodiments, assessing efficacy includes measuring cell death and/or viability of the immortalized cells.

In some embodiments, the measurement includes performing a cell viability assay. In some embodiments, the measurement includes performing a cell cytotoxicity assay. In some embodiments, the measurement is a real-time cell viability assay, ATP cell viability assay, live cell protease viability assay, tetrazolium reduced cell viability assay, resazurin reduced cell viability assay, apoptotic cell viability assay. and performing an assay selected from a dead-cell protease release cytotoxicity assay, a lactate dehydrogenase release cytotoxicity assay, and a DNA dye cytotoxicity assay.

A further aspect of the disclosure provides a method of assessing the potency of tumor infiltrating lymphocytes (TILs), comprising co-culturing a clonal population of TILs and engineered cancer cells, wherein the engineered cancer cells are anti- expressing a CD3 antibody or anti-CD3 antibody fragment and assessing killing and/or viability of the engineered cancer cells.

In some embodiments, the TIL expresses CD3 antigen.

Still another aspect of the disclosure provides a method of assessing the potency of polyclonal T cells, comprising co-culturing a clonal population of polyclonal T cells and engineered cancer cells, comprising: cancer cells expressing an anti-CD3 antibody or anti-CD3 antibody fragment, and assessing death and/or viability of the engineered cancer cells.

In some embodiments, the polyclonal T cells express CD3 antigen.

In some embodiments, the engineered cancer cells express anti-CD3 antibody fragments. For example, the anti-CD3 antibody fragment may be an anti-CD3 single chain variable fragment (scFv). In some embodiments, the anti-CD3 scFv is mOKT3 scFv. In some embodiments, the mOKT3 scFv is a low affinity mOKT3 scFv variant.

In some embodiments, the low affinity mOKT3 scFv variant binds CD3 with a lower KD than the dissociation constant (KD) of mOKT3 scFv, wherein the KD of mOKT3 scFv is about 5×10 ^-10 M. In some embodiments, the low affinity mOKT3 scFv variant binds CD3 with a KD that is at least 1000-fold lower than the dissociation constant KD of the mOKT3 scFv. In some embodiments, the low affinity mOKT3 scFv variant comprises an amino acid sequence with the R55L and Y57T mutations relative to the amino acid sequence of SEQ ID NO:2.

In some embodiments, the anti-CD3 antibody or anti-CD3 antibody fragment is a membrane-tethered anti-CD3 antibody or a membrane tethered anti-CD3 antibody fragment, respectively.

In some embodiments, the TIL is an engineered TIL (eTIL). In some embodiments, the eTIL is an edited eTIL. In some embodiments, the edited eTIL includes genomic modifications.

In some embodiments, the engineered cancer cells are selected from engineered melanoma cells, engineered colorectal cancer cells, engineered cholangiocarcinoma cells, and engineered breast cancer cells. In some embodiments, the engineered cancer cells are engineered melanoma cells.

In some embodiments, the co-culture is for at least 24 hours, such as about 24 to 72 hours.

In some embodiments, the measurement is a real-time cell viability assay, ATP cell viability assay, live cell protease viability assay, tetrazolium reduced cell viability assay, resazurin reduced cell viability assay, dead cell protease release cytotoxicity assay, rock and performing an assay selected from a tate dehydrogenase release cytotoxicity assay and a DNA dye cytotoxicity assay.

A further aspect of the disclosure provides a method of assessing the potency of a TIL, comprising co-culturing a TIL, an immortalized cell, and a bispecific molecule that activates the T cell and binds to the immortalized cell, and determining the potency of the TIL. Includes an evaluation step. In some embodiments, the TIL expresses CD3 antigen. In some embodiments, the bispecific molecule includes a molecule that binds CD3.

In some embodiments, the TIL is an engineered TIL (eTIL). In some embodiments, the eTIL is an edited eTIL. In some embodiments, the edited eTIL includes genomic modifications.

Another aspect of the disclosure provides a method of assessing the potency of polyclonal T cells, comprising co-producing polyclonal T cells, immortalized cells, and a bispecific molecule that activates the T cells and binds to the immortalized cells. It includes steps of culturing and evaluating the potency of polyclonal T cells. In some embodiments, the polyclonal T cells express CD3 antigen. In some embodiments, the bispecific molecule includes a molecule that binds CD3.

In some embodiments, the immortalized cells comprise a genetic population of immortalized cells. In some embodiments, the immortalized cells are human cells. In some embodiments, the immortalized cells are (e.g., human) cancer cells. In some embodiments, the cancer cells are selected from melanoma cells, colorectal cancer cells, cholangiocarcinoma cells, and breast cancer cells.

In some embodiments, the bispecific molecule includes a molecule that binds CD19. In some embodiments, the bispecific molecule includes a molecule that binds CD3. In some embodiments, the bispecific molecule includes a molecule that binds CD19 and CD3.

In some embodiments, the bispecific molecule is CD19-CD3 BiTE®.

In some embodiments, the co-culture is for at least 24 hours, such as about 24 to 72 hours.

In some embodiments, the measurement includes performing a cell viability assay. In some embodiments, the measurement includes performing a cytotoxicity assay. In some embodiments, the measurement is a real-time cell viability assay, ATP cell viability assay, protease viability assay, tetrazolium reduced cell viability assay, resazurin reduced cell viability assay, dead cell protease release cytotoxicity assay, lactate and performing an assay selected from a dehydrogenase release cytotoxicity assay and a DNA dye cytotoxicity assay.

These and other features and advantages of the present disclosure will be more fully understood from the following detailed description of exemplary embodiments taken in conjunction with the accompanying drawings.
Figure 1 shows A375 cells transduced with 0 μl, 25 μl, 50 μl, 100 μl, 200 μl, 400 μl, or 800 μl of pKSQ366 or pKSQ367 lentivirus encoding mOKT3, co-cultured with human Pan-CD3 ⁺ T cells overnight. A drawing showing what has been done. The next day, CD69 activation was assessed by flow cytometry. A375 cells transduced with various amounts of pKSQ366 virus were found to induce similar levels of CD69 activation, except for 25 μl of virus. A375 cells transduced with pKSQ367 induced slightly higher levels of CD69 activation, and similar levels of activation were found using various amounts of virus.
Figure 2 shows mOKT3 surface expression confirmed by staining A375-pKSQ367 and non-transduced cells with a goat-anti-mouse antibody. We found that 99.2% of A375-pKSQ367 expressed mOKT3.
Figures 3A - 3C depict pre-REP TILs co-cultured with A375-pKSQ367 for 3 days at various effector to target cell ratios (E:T). No killing of A375 by pre-REP TILs was observed except at 10:1 in D2777. Increased TIL recognition and subsequent killing of A375-pKSQ367 are observed in all E:T.
Figure 4 shows that constructs with various membrane anchors, signal peptides and scFv linkers all produce robust activation of T cells. K562 cells with the indicated constructs were co-cultured with pan T cells. After 24 hours, T cells were stained for CD8 and CD69 surface expression and measured by flow cytometry. CD69 expression was high after coculture with K562 cells expressing mOKT3, regardless of the anchor used to tether to the plasma membrane of K562 cells.
Figure 5 shows that high affinity A375 pKSQ367 shows high expression of the mOKT3 marker compared to the A375 parental cell line from which it is derived.
Figure 6 shows that low-affinity cell line A375 pKSQ397 shows high mOKT3 marker expression. Increasing the volume of virus used for transduction did not increase mOKT3 expression on the surface of these cells.
Figure 7 shows that low affinity cell line A375 pKSQ398 shows intermediate level of mOKT3 marker expression. Increasing the volume of virus used for transduction did not increase mOKT3 expression on the surface of these cells.
Figure 8 shows that pan T cells show increased expression of CD69 on the cell surface after co-culture with A375 pKSQ367 target cells. This indicates recognition of target cells (A375 pSKQ367) and activation of pan T cells. In contrast, pan T cells show little CD69 expression after coculture with A375 blast cells. This indicates that activation of pan T cells does not increase due to lack of recognition. As a control, pan T cells cultured alone show a lack of basal CD69 expression.
Figure 9 shows that pan T cells show little expression of CD69 on the cell surface after co-culture with A375 pKSQ396 target cells. This expression level is comparable to pan T cells cocultured with A375 parental cells ( Fig. 8 ). This indicates that there is no increase in activation due to lack of target cell recognition by pan T cells.
Figure 10 is a diagram showing the expression of CD69 on the cell surface of pan T cells after co-culture with A375 pKSQ397 target cells. This expression level indicates recognition of target cells (A375 pSKQ397) and activation of pan T cells. Target cells transduced with larger volumes of virus appear to have the ability to slightly increase the activation of pan T cells.
Figure 11 is a diagram showing the expression of CD69 on the cell surface of pan T cells after co-culture with A375 pKSQ398 target cells. This expression level indicates recognition of target cells (A375 pSKQ398) and activation of pan T cells. Target cells transduced with larger volumes of virus appear to have the ability to slightly increase the activation of pan T cells.
Figure 12 depicts monolayer co-culture of A375 parental cells with gene target-edited TILs (no EP, OLFR , SOCS1 , CBLB ) from TIL donor 3239. A reduction or no difference in target A375 cell growth was observed due to lack of mOKT3 surface expression.
Figure 13 shows monolayer co-culture of A375 pKSQ367 cells with gene target-edited TILs (no EP, OLFR , SOCS1 , CBLB ) from TIL donor 3239. Due to the previously observed high levels of mOKT3 surface expression, a significant reduction in target A375 pKSQ367 cell growth was observed.
Figure 14 shows monolayer co-culture of A375 pKSQ398 cells with gene target-edited TIL (no EP, OLFR , SOCS1 , CBLB ) from TIL donor 3239. Because pKSQ398 has a lower affinity for CD3 compared to pKSQ367, there was a decrease in the kinetics of TIL-kill of target A375 pKSQ398 cells. All groups have similar activity against cell lines, suggesting that low OKT3 affinity makes it difficult for TILs to kill their targets.
Figure 15 is a diagram showing spheroid morphology observed by plating 10,000 A375-pKSQ367 or A375-pKSQ398 on an ultra-low attachment plate and imaging over 10 days.
Figure 16 shows 10,000 A375-pKSQ367 or A375-pKSQ398 plated on an ultra-low adhesion plate. Non-electroporated (no EP) and SOCS1 edited TILs were added at various E:T ratios after 4 days. Imaging was performed continuously throughout the spheroid formation and co-culture period. These images were collected after 36 hours for A375-pKSQ367 and after 72 hours for A375-pKSQ398.
Figure 17 shows 10,000 A375-pKSQ367 or A375-pKSQ398 plated on an ultra-low adhesion plate. Non-electroporated (no EP) and SOCS1 edited TILs were added at various E:T ratios after 4 days. Imaging was performed continuously throughout the spheroid formation and co-culture period. These images were collected after 72 hours of co-culture.
Figure 18 is a diagram showing 10,000 A375-pKSQ367 plated on an ultra-low adhesion plate. Non-electroporated (no EP) and SOCS1 edited TILs were added at various E:T ratios after 4 days. Imaging was performed continuously throughout the spheroid formation and co-culture period. These images were collected after 5 days of co-culture.
Figure 19 is a diagram showing 5,000 or 10,000 A375-pKSQ398 plated on an ultra-low adhesion plate. Non-electroporated (no EP) and SOCS1 edited TILs were added at various E:T ratios after 4 days. Imaging was performed continuously during spheroid formation and co-culture. These images were collected after 5 days of co-culture.
Figure 20 depicts the cytotoxicity of SOCS1 edited TILs and control unedited TILs on A375-OKT3lt spheroids over 72 hours at different effector:target (E:T) ratios. In monolayer cultures, SOCS1 edited cells did not differ from unedited cells in the killing of both A375-pKSQA367 and A375-pKSQ368 cells ( Figures 13 and 14 ). In a 3D spheroid setting, SOCS1 edited cells showed enhanced cytotoxicity compared to controls. This suggests that the sensitivity of the assay is different in the spheroid setting.
Figure 21 depicts the IFNγ produced by SOCS1 edited TILs and control unedited TILs on A375-OKT3lt spheroids at different effector:target (E:T) ratios at 24 hours.
Figure 22 depicts IL-6 produced by SOCS1 edited TIL and control unedited TIL on A375-OKT3lt spheroids at different effector:target (E:T) ratios at 24 hours.
Figure 23 shows the level of luminescence detected in the supernatant after 24 hours of TIL-spheroid co-culture.
Figure 24 shows LDH levels calculated in the supernatant after 24 hours of TIL-spheroid co-culture.

The present disclosure provides, in some aspects, methods and compositions useful for measuring the antitumor potency of tumor infiltrating lymphocytes (TILs). In some embodiments, these methods include, for example, co-culturing a TIL and an immortalized cell and assessing TIL potency using an approach comprising co-culturing a TIL, an immortalized cell, and a bispecific molecule. do.

Many TIL potency assays currently used to monitor T cell function focus on the use of cytokine (e.g., IFN-γ or IL-2) release assays. For example, T cell activation can be determined by measuring IFN-γ secretion after a brief co-culture period with non-cell based T cell activation reagents, including anti-CD3/anti-CD28 antibody coated beads. Because cytokines such as IFN-γ enhance MHC I and Fas expression on target cells, cytokine secretion is correlated with cytolytic activity of CD8 ⁺ T cells. For example, a TIL may be considered potent if interferon gamma (IFN-γ) release is greater than 50 pg/ml, greater than 100 pg/ml, greater than 150 pg/ml, or greater than 200 pg/ml upon TCR stimulation.

However, their direct activation by target tumor cells requires that TILs must be able to at least interact with the target cells and produce/release mediators for inducing death, such as degranulation of cytolytic granules containing granzyme B and perforin. Killing is an equally important indicator of clinical efficacy that is not measured in currently available cytokine release assays induced by non-cell-based TIL activation methods. TIL potency assays provided herein may be used to determine apoptosis and/or viability or degranulation, such as CD107a, of target cells (e.g., immortalized cells, e.g., cancer cells) in a more physiologically relevant TIL/tumor cell co-culture setting. Measurement of surrogate markers of inflammation or the opportunity to directly measure the production of effector cytokines/chemokines (e.g., pro-inflammatory cytokines/chemokines) such as IFN-γ, IL-6, IL_2, and TNFα. Activating TILs through tumor cell-based interactions improves existing TIL potency assays (e.g., cytokine release assays).

“TIL potency assay” refers to an assay used to characterize, e.g., quantify, the antitumor activity of TILs (e.g., cytokine production, TIL degranulation, tumor growth inhibition). TIL potency assays can be used to assess TIL antitumor activity before and/or after rapid expansion of TILs and prior to application for clinical use, such as adoptive cell therapy (ACT).

Tumor infiltrating lymphocytes

Engineered TILs and/or tumor infiltrating lymphocytes (TILs) comprising edited TILs can be characterized based on the potency of their antitumor activity (e.g., inhibition of tumor cell growth).

The phrase “tumor infiltrating lymphocytes” or “TIL” refers to a population of lymphocytes that have left the subject's bloodstream and migrated to the tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells, CD4+ T cells, such as Th1 and Th17 CD4+ T cells, natural killer T cells, and natural killer (NK) cells. TIL includes both primary and secondary TIL. “Primary TILs” are those obtained from patient tissue samples (sometimes referred to as “freshly harvested”) as outlined herein, and “secondary TILs” are bulk TILs and expanded TILs (“REP TILs” or “REP TILs”). Any expanded or proliferated TIL cell population, including but not limited to "post-REP TIL"). In some embodiments, the primary TIL comprises tumor-reactive T cells obtained from the patient's peripheral blood. The TIL cell population may include genetically modified or otherwise engineered TILs. “TIL” also refers to a population of lymphocytes that leave a subject's bloodstream, travel to a tumor, and then set out to re-enter the bloodstream.

As generally outlined herein, TILs are generally harvested from patient samples and manipulated to expand their numbers prior to implantation into the patient. In some embodiments, TILs may be genetically engineered as discussed below. Typically, TILs are initially obtained from patient tumor samples (“primary TILs”) and then expanded into larger populations for further manipulation as described herein, optionally cryopreserved and restimulated, and optionally As an indication of TILs, phenotypic and metabolic parameters are evaluated.

The term “subject” refers to a human. In some embodiments, such human may be a patient in need of immunotherapy comprising an expanded population of the patient's own TILs. In another embodiment, this human may be another patient in need of immunotherapy comprising an expanded population of the patient's own TILs.

TILs can generally be defined biochemically using cell surface markers or functionally by their ability to infiltrate tumors and influence treatment. TILs can generally be classified as expressing one or more of the following biomarkers: CD4, CD8, TCRαβ, TCRgd, CD27, CD28, CD56, CCR7, CD45RA, CD45RO, CD95, PD-1, and CD25. Additionally, alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors when reintroduced to the patient.

Adoptive cell therapy using TILs cultured ex vivo by conventional TIL preparation processes involves at least two steps: a pre-REP step followed by at least one rapid expansion protocol (REP) step. Adoptive cell therapy resulted in successful therapy after host immunosuppression in melanoma patients. Current injection tolerance parameters depend on the composition readout of the TIL (e.g., CD28, CD8, or CD4 positivity) and the numerical fold of the expansion and viability of the REP product.

The phrase “population of cells” or “population of TILs” refers to a number of cells or TILs that share common traits. In general, populations typically vary from 1×10 ⁶ to 1×10 ¹⁰ , with different TIL populations containing different numbers. For example, initial growth of primary TILs in the presence of IL-2 can result in a population of bulk TILs of approximately 1× ¹⁰ cells. REP expansion is typically performed to provide a population of 1.5×10 ⁹ to 1.5×10 ¹⁰ cells for injection. In some embodiments, the population of cells is monoclonal. In another embodiment, the population of cells is polyclonal. In some embodiments, when a population of cells is polyclonal, the cells still share one or more common traits. Monoclonal T cell populations will result in a predominance of single TCR-gene rearrangement patterns. In contrast, polyclonal T cell populations have different TCR-gene rearrangement patterns, which may make them more effective in certain situations.

In some embodiments, the TIL is a high affinity T cell receptor (TCR), e.g., a TCR targeting a tumor-associated antigen, such as MAGE-1, HER2 or NY-ESO-1, or a tumor-associated cell surface molecule. Genetically engineered to contain additional functions, including but not limited to chimeric antigen receptors (CARs) that bind to lineage-restricted cell surface molecules (e.g., mesothelin) or lineage-restricted cell surface molecules (e.g., EGFR, CD19, or HER2) do.

The term “engineered TIL” or “eTIL” refers to one or more TILs as well as TILs comprising one or more genomic modifications achieved through non-natural means that result in reduced expression and/or function of one or more endogenous target genes. Includes TILs that contain non-naturally occurring gene-regulatory systems that can reduce the expression and/or function of endogenous target genes. “Unmodified TIL” or “control TIL” means that the genome has not been modified through non-naturally occurring means and does not contain a non-naturally occurring gene-regulatory system or a control gene-regulatory system (e.g., an empty vector control, refers to a TIL or group of TILs containing non-targeting gRNA, scrambled siRNA, etc.). Naturally occurring TILs that have reduced expression and/or function of one or more endogenous genes are included under the term unmodified or control TIL.

In some embodiments, engineered TILs produced by the methods described herein include one or more modifications (e.g., insertions, deletions, or mutations of one or more nucleic acids) within the genomic DNA sequence of the endogenous target gene. Resulting in reduced expression and/or function. In some embodiments, modifications in the genomic DNA sequence reduce or inhibit mRNA transcription, resulting in reduced expression levels of encoded mRNA transcripts and proteins. In some embodiments, modifications in the genomic DNA sequence reduce or inhibit mRNA translation, resulting in reduced expression levels of the encoded protein. In some embodiments, the modification in the genomic DNA sequence results in reduced or altered function compared to an unmodified (i.e., wild-type) version of the endogenous protein (e.g., a dominant-negative mutant described below). encodes a modified endogenous protein with

In some embodiments, the modified TIL further comprises an engineered antigen-specific receptor that recognizes a protein target expressed by a target cell, such as a tumor cell or antigen presenting cell (APC). The term “engineered antigen receptor” refers to a non-naturally occurring antigen-specific receptor, such as a chimeric antigen receptor (CAR) or recombinant T cell receptor (TCR). In some embodiments, the engineered antigen receptor is a CAR comprising an extracellular antigen binding domain fused to a cytoplasmic domain comprising a signaling domain through hinge and transmembrane domains. In some embodiments, the CAR extracellular domain binds an antigen expressed by the target cell in an MHC-independent manner, which leads to activation and proliferation of the RE cell. In some embodiments, the extracellular domain of the CAR recognizes a tag fused to an antibody or antigen-binding fragment thereof. In this embodiment, the antigen-specificity of the CAR depends on the antigen-specificity of the labeled antibody, allowing a single CAR construct to be used to target multiple different antigens by substituting one antibody for another. In some embodiments, the extracellular domain of the CAR may comprise an antigen-binding fragment derived from an antibody. Antigen binding domains useful in the present disclosure include, for example, scFv; antibodies; the antigen binding region of an antibody; Variable regions of heavy/light chains; and single chain antibodies.

In some embodiments, the intracellular signaling domain of the CAR may be derived from the TCR complex zeta chain (e.g., CD3ξ signaling domain), FcγRIII, FcεRI, or T-lymphocyte activation domain. In some embodiments, the intracellular signaling domain of the CAR further comprises a costimulatory domain, e.g., a 4-1BB, CD28, CD40, MyD88 or CD70 domain. In some embodiments, the intracellular signaling domain of the CAR further comprises any two of two costimulatory domains, e.g., 4-1BB, CD28, CD40, MyD88, or CD70 domains. Exemplary CAR structures and intracellular signaling domains are known in the art (e.g., WO 2009/091826; US 20130287748; WO 2015/142675; WO 2014/055657; and WO 2014/055657, incorporated herein by reference) 2015/090229).

CARs specific for various tumor antigens are known in the art, for example, CD171-specific CAR (Park et al ., Mol Ther (2007) 15(4):825-833), EGFRvIII- Specific CAR (Morgan et al ., Hum Gene Ther (2012) 23(10):1043-1053), EGF-R-specific CAR (Kobold et al ., J Natl Cancer Inst (2014) 107(1):364]), carbonic anhydrase K-specific CAR (Lamers et al ., Biochem Soc Trans (2016) 44(3):951-959]), FR-α-specific CAR (Kershaw et al., Clin Cancer Res (2006) 12(20):6106-6015), HER2-specific CAR (Ahmed et al ., J Clin Oncol (2015) 33(15)1688- 1696; Nakazawa et al ., Mol Ther (2011) 19(12):2133-2143; Ahmed et al ., Mol Ther (2009) 17(10):1779-1787; Cell Res (2016) 26(7):850-853; Morgan et al ., Mol Ther (2010) 18(4):843-851; Grada et al ., Mol Ther Nucleic Acids (2013) 9(2):32]), CEA -specific CAR (Katz et al ., Clin Cancer Res (2015) 21(14):3149-3159), IL13Rα2-specific CAR (Brown et al ., Clin Cacner Res (2015) 21( 18):4062-4072]), GD2-specific CAR (Louis et al ., Blood (2011) 118(23):6050-6056; Caruana et al ., Nat Med (2015) 21(5): 524-529]), ErbB2-specific CAR (Wilkie et al ., J Clin Immunol (2012) 32(5):1059-1070]), VEGF-R-specific CAR (Chinnasamy et al . , Cancer Res (2016) 22(2):436-447]), FAP-specific CAR (Wang et al ., Cancer Immunol Res (2014) 2(2):154-166]), MSLN-specific enemy CAR (Moon et al , Clin Cancer Res (2011) 17(14):4719-30), NKG2D-specific CAR (VanSeggelen et al , Mol Ther (2015) 23(10):1600 -1610]), CD19-specific CARs (Axicabtagene ciloleucel (Yescarta®) and Tisagenlecleucel (Kymriah®). Please also refer to the literature [82/337 Li et al ., J Hematol and Oncol (2018) 11(22)] for a review of clinical trials of tumor-specific CAR.

As generally outlined herein, TILs are generally harvested from patient samples and manipulated to expand their numbers prior to implantation into the patient. In some embodiments, TILs may be genetically engineered as discussed below. Typically, TILs are initially obtained from patient tumor samples (“primary TILs”) and then expanded into larger populations for further manipulation, optionally cryopreserved and restimulated, and optionally phenotypic and metabolic as an indication of the TIL. It is evaluated against parameters.

Patient tumor samples can be obtained using methods known in the art, generally through surgical resection, needle biopsy, or other means to obtain samples containing a mixture of tumor and TIL cells. In general, tumor samples can be from any solid tumor, including primary tumors, invasive tumors, or metastases. Solid tumors include bladder, brain, breast (including triple-negative breast), cervix, colorectal, stomach, endometrial, kidney, lip and oral cavity, and head and neck cancers (e.g., head and neck squamous cell carcinoma (HNSCC)). blastoma, glioblastoma multiforme, neuroblastoma, liver cancer, mesothelioma, lung cancer (including non-small cell lung cancer (NSCLC) and small cell lung cancer) skin cancer (including but not limited to squamous cell carcinoma, basal cell carcinoma, non-melanoma skin cancer, and melanoma) (not limited to), ovarian cancer, uveal cancer, uterine cancer, pancreatic cancer, prostate cancer, sarcoma, and thyroid cancer. In some embodiments, useful TILs are obtained from malignant melanoma tumors because they have been reported to have particularly high levels of TILs. TILs can be obtained using primary lung (non-small cell lung cancer (NSCLC)), bladder, cervical, and melanoma tumors or their metastases.

Once obtained, the tumor sample is fragmented using sharp dissection into small pieces, typically about 1 to about 8 mm3 or about 0.5 to about 4 mm3, with about 2 to 3 mm3 being particularly useful. TILs are cultured from these fragments using enzymatic tumor digests. These tumor digests were cultured in enzyme medium (e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2mM glutamate, 10 μg/ml gentamicin, 30 units/ml DNase, and 1.0 mg/ml collagenase). ), followed by mechanical dissociation (e.g., using a tissue dissociator). Tumor digests were prepared by placing the tumor in enzyme medium, mechanically dissociating the tumor for approximately 1 minute, and then incubating at 37°C, 5% CO ₂ for 30 minutes, followed by mechanical dissociation and incubation under these conditions until only small tissue fragments were present. It can be produced by repeating the cycle. Upon completion of this process, if the cell suspension contains significant numbers of red blood cells or dead cells, these cells can be removed by performing density gradient separation using FICOLL branched hydrophilic polysaccharide. Alternative methods known in the art may be used, such as those described in US Patent Publication No. 2012/0244133 A1, the disclosure of which is incorporated herein by reference in its entirety. Any of the above methods can be used in any of the embodiments described herein for methods of expanding TILs or methods of treating cancer.

Typically, the harvested cell suspension is referred to as the “primary cell population” or “freshly harvested” cell population. In some embodiments, fragmentation includes physical fragmentation, including, for example, decomposition and digestion. In some embodiments, fragmentation is physical fragmentation. In some embodiments, fragmentation is incision. In some embodiments, fragmentation is by digestion. In some embodiments, TILs may initially be cultured from enzymatic tumor digests and tumor fragments obtained from patients.

In some embodiments, TILs are obtained from tumor digests. In some embodiments, the tumor digest is a mechanically dissociated tumor in an enzyme medium, such as, but not limited to, RPMI 1640, 2mM GlutaMAX, 10 mg/ml gentamicin, 30 U/ml DNase, and 1.0 mg/ml collagenase. produced by incubation, followed by mechanical dissociation (GentleMACS, Miltenii Biotech, Auburn, CA, USA). In some embodiments, mechanically dissociated tumors will be broken into approximately 1 mm 3 pieces. After placing the tumor in enzyme medium, the tumor can be mechanically dissociated for approximately 1 minute. The solution can then be incubated at 37°C, 5% CO ₂ for 30 minutes and then mechanically disrupted again for about 1 minute. After another incubation at 37°C, 5% CO ₂ for 30 minutes, the tumor can be mechanically disrupted a third time for approximately 1 minute. In some embodiments, after the third mechanical disruption if large pieces of tissue are present, one or two additional mechanical dissociations may be applied to the sample with or without an additional incubation of 30 minutes at 37°C in 5% CO ₂ . In some embodiments, upon completion of the final incubation, if the cell suspension contains significant numbers of red blood cells or dead cells, density gradient separation can be performed using FICOLL to remove these cells.

In some embodiments, cells may optionally be frozen or cryopreserved after sample collection and stored frozen prior to entering the expansion phase.

In some embodiments, the TILs are derived from initial tumor fragmentation or non-aggregation up to a total of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, Extends for 26, 27 or 28 days. In some embodiments, the TIL extends for a total of 9 to 25 days, 9 to 21 days, or 9 to 14 days. In some embodiments, the TIL is extended for up to a total of 9 days. In some embodiments, the TIL extends for up to a total of 10 days. In some embodiments, the TIL extends for up to a total of 11 days. In some embodiments, the TIL extends for up to a total of 12 days. In some embodiments, the TIL extends for up to a total of 13 days. In some embodiments, the TIL is extended for up to a total of 14 days. In some embodiments, the TIL is extended for up to a total of 15 days. In some embodiments, the TIL is extended for up to a total of 16 days. In some embodiments, the TIL extends for up to a total of 17 days. In some embodiments, the TIL extends for up to a total of 18 days. In some embodiments, the TIL extends for up to a total of 19 days. In some embodiments, the TIL extends for up to a total of 20 days. In some embodiments, the TIL extends for up to a total of 21 days. In some embodiments, the TIL extends for up to a total of 22 days. In some embodiments, the TIL extends for up to a total of 23 days. In some embodiments, the TIL extends for up to a total of 24 days. In some embodiments, the TIL extends for up to a total of 25 days. In some embodiments, the TIL extends for up to a total of 26 days. In some embodiments, the TIL extends for up to a total of 27 days. In some embodiments, the TIL extends for up to a total of 28 days.

In some embodiments, expanded TILs are analyzed for expression of multiple phenotypic markers, including those described herein. In some embodiments, the marker is selected from: TCRα/β, CD57, CD28, CD4, CD27, CD56, CD8a, CD45RA, CD45RO, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some embodiments, the expression of one or more regulatory markers is measured from the following group: CD137, CD8a, Lag3, CD4, CD3, PD-1, TIM-3, CD69, CD8a, TIGIT, CD4, CD3, KLRG1. and CD154.

In some embodiments, the memory marker is CCR7 or CD62L. In some embodiments, restimulated TILs are assessed for cytokine release using a cytokine release assay. In some embodiments, TILs are assessed for interferon-gamma (IFN-γ) secretion in response to stimulation with OKT3 or co-culture with autologous tumor digest. In some embodiments, TILs are assessed for IL-6 secretion in response to stimulation with OKT3 or co-culture with autologous tumor digest. Additional effector cytokines that can be measured include IL-1, IL-2, IL-12, IL-17, IL-18, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNFα). Including but not limited to these. Chemokines such as CXCL10, CXCL13, CCL1, CCL3, CCL4, CCL5, CCL9/10, CCL17, CCL22, CCL23 and XCL1 can also be assessed.

TILs are assessed for various regulatory markers such as TCRα/β, CD56, CD27, CD28, CD57, CD45RA, CD45RO, CD25, CD127, CD95, IL-2R, CCR7, CD62L, KLRG1 and CD122.

immortalized cells

Immortalized cells are cells that have been engineered to proliferate indefinitely and can be cultured for long periods of time. Immortalized cell lines typically come from a variety of sources with mutations that allow them to continue dividing, such as chromosomal abnormalities or tumors. Immortalized cells are therefore considered “engineered” cells. The immortalized cell population may be a heterogeneous population or may be derived from a single immortalized clone (to form a monogenic population). In some embodiments, the immortalized cells comprise a heterogeneous population of immortalized cells. In some embodiments, the immortalized cells comprise a genetic population of immortalized cells.

Immortalized cells may be derived from a variety of species and/or origins. For example, the immortalized cells can be immortalized animal cells, immortalized human cells, or combinations thereof. In some embodiments, the immortalized cells are immortalized human cells.

In some embodiments, the immortalized cells are cancer cells. For example, cancer cells may include, but are not limited to, melanoma cells, colorectal cancer cells, cholangiocarcinoma cells, and breast cancer cells. In some embodiments, the cancer cells are selected from melanoma cells, colorectal cancer cells, cholangiocarcinoma cells, and breast cancer cells.

Immortalized cells that can be engineered for use in the TIL potency assays described herein include A375 melanoma cells (e.g., ATCC® CRL-1619™), K562 pluripotent, hematopoietic malignant cells, primary cells (e.g. , ATCC® CCL-243™), human embryonic kidney (HEK) 293T cells (e.g., ATCC® CRL-1573), and Chinese hamster ovary (CHO) cells (e.g., ATCC® CCL-61™). You can, but are not limited to these. In some embodiments, the immortalized cells are selected from A375 cells, K562 cells, primary cells, HEK293T cells, and CHO cells. It should be understood that immortalized cells useful in the assays and methods described herein are not limited to the examples described above. Other immortalized cell lines are known in the art and can be used according to the present disclosure.

The use of cell lines expressing immunosuppressive markers that suppress T cell responses, such as PD-L1, PD-L2, to test whether TILs are resistant to inhibitory signals is also contemplated herein. In some embodiments, immortalized cells that can be engineered for use in TIL potency assays include cell lines that express immunosuppressive markers that suppress T cell responses, such as PD-L1.

Cell lines expressing costimulatory markers such as CD80/86, OX40L and/or 41BBL to enhance T cell responses can also be engineered for use in TIL potency assays. In some embodiments, immortalized cells that can be engineered for TIL potency assays include cell lines capable of expressing costimulatory markers, such as CD80/86, to enhance T cell responses.

The phrase “tumor cell” or “cancer cell” refers to a cell that divides in an uncontrolled manner to form a solid tumor or flood the blood with abnormal cells. Healthy cells stop dividing when they no longer need more daughter cells, but tumor cells, or cancer cells, continue to produce copies. They can also spread from one part of the body to another in a process known as metastasis. Tumor cells include bladder, brain, breast (including triple-negative breast), cervix, colorectal, stomach, endometrial, kidney, lip and oral cavity, and head and neck cancers (e.g., head and neck squamous cell carcinoma (HNSCC)). blastoma, glioblastoma multiforme, neuroblastoma, liver cancer, mesothelioma, lung cancer (including non-small cell lung cancer (NSCLC) and small cell lung cancer) skin cancer (including but not limited to squamous cell carcinoma, basal cell carcinoma, non-melanoma skin cancer, and melanoma) can be isolated from a number of cancer types, including ovarian cancer, uveal cancer, uterine cancer, pancreatic cancer, prostate cancer, sarcoma, and thyroid cancer. In some embodiments, cancer cells are also isolated from lymphoma. Tumor cells can be isolated from primary tumors and metastases.

Immortalized cells are described throughout in the context of co-culture with TILs, but immortalized cells are any tumor cell, such as a cancer cell, that expresses or has been engineered to express molecules that activate T cells, e.g., that bind to T cell antigens. It must be understood that it can be replaced with .

Molecules that activate lymphocytes

The engineered immortalized cells of the present disclosure may contain or express molecules that activate T cells.

“Molecules that activate lymphocytes” and “molecules that activate T cells” refer to non-endogenous stimuli that activate cells. In the endogenous process, T cells are activated when peptide antigens are presented, for example, by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). When activated, T cells divide rapidly and secrete cytokines that regulate or assist immune responses. The endogenous T cell activation process involves at least (a) activation of a TCR complex containing CD3, and (b) costimulation of CD28 or 4-1BB by proteins on the APC surface. It is known in the art that endogenous activation of T cells can be simulated by stimulation of T cells with CD3, CD28, or 4-1BB agonists (e.g., antibodies). Therefore, CD3, CD28 and/or 4-1BB can together activate T cells.

Activated T cells increase in number or proliferate and begin producing cytokines (activated TILs) to enhance the immune response.

Immortalized cells may contain and/or express molecules that activate T cells by binding (e.g., directly binding) to T cell antigens. In some embodiments, the TILs (e.g., engineered TILs and/or edited TILs) express a T cell antigen. Non-limiting examples of T cell antigens include CD3, CD28, CD2, 41BB, OX40, GITR, ICOS, CD4, CD8. In some embodiments, the T cell antigen is CD3. In some embodiments, the T cell antigen is CD28. In some embodiments, the T cell antigen is CD2.

The term “CD3” refers to the CD3 (cluster of differentiation 3) T cell coreceptor, which helps activate both cytotoxic T cells (CD8+ naive T cells) and also T helper cells (CD4+ naive T cells). CD3 is a protein complex composed of six distinct polypeptide chains (two CD3 zeta chains, two CD3 epsilon chains, one CD3e gamma chain, and one CD3 delta chain). These chains associate with the T cell receptor (TCR) alpha and beta chains (or gamma and delta chains) to generate activation signals in T lymphocytes. The TCR alpha and beta chains (or gamma and delta chains) and CD3 molecules together make up the TCR complex. The human CD3E gene is identified by the National Center for Biotechnology Information (NCBI) gene ID 916. An exemplary nucleotide sequence for the human CD3E gene is NCBI Reference Sequence: NG_007383.1.

The term “CD28” refers to cluster of differentiation 28, one of the proteins expressed on T cells that provide costimulatory signals necessary for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation through CD28 can provide a powerful signal for the production of various cytokines such as interleukins. CD28 is the receptor for the CD80 and CD86 proteins. When activated by Toll-like receptor ligands, CD80 expression is upregulated on antigen presenting cells (APCs). The human CD28 gene is identified by NCBI gene ID 940. An exemplary nucleotide sequence for the human CD28 gene is NCBI Reference Sequence: NG_029618.1. An exemplary amino acid sequence of a human CD28 polypeptide is as follows:

MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRR PGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 19).

The term “CD2” refers to cluster of differentiation 2, a cell adhesion molecule found on the surface of T cells and natural killer (NK) cells. CD2 interacts with other adhesion molecules and acts as a costimulatory molecule in T cells and NK cells. The human CD2 gene is identified by NCBI gene ID 914. An exemplary nucleotide sequence for the human CD2 gene is NCBI Reference Sequence: NG_050908.1. An exemplary amino acid sequence of a human CD2 polypeptide is as follows:

MSFPCKFVASFLLIFNVSSKGAVSKEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSC PEKGLDIYLIIGICGGGSLLMVFVALLVFYITKRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO: 20).

In some embodiments, the immortalized cells comprise molecules that activate T cells by binding, e.g., specifically binding to a T cell antigen. In some embodiments, the immortalized cells comprise molecules that activate T cells by binding to CD3 antigen. In some embodiments, the immortalized cells comprise molecules that activate T cells by binding to CD28 antigen. In some embodiments, the immortalized cells comprise molecules that activate T cells by binding to CD2 antigen.

The phrase “specifically binds” refers to a molecule (e.g., an antibody) that interacts with a particular antigen (e.g., a T cell antigen) with high specificity when compared to another antigen for which the complex has a lower binding affinity. refers to Specific binding interactions may be mediated through ionic bonds, hydrogen bonds, or other types of chemical or physical associations. In some embodiments, a molecule specifically binds to a particular antigen when it recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In some embodiments, the molecule that activates the T cell is less than about 10 ^-5 M, such as less than about 10 ^-6 M, 10 ^-7 M, 10 ^-8 M, 10 ^-9 M or 10 ^-10 M or lower. Binds to T cell antigens with high affinity (KD).

In some embodiments, the molecule that activates T cells is a T cell agonist. The term “agonist” refers to a chemical, molecule, macromolecule, molecular complex, or macromolecular complex that binds to a target on the cell surface or in soluble form. In certain embodiments, when an agonist binds to a target on the cell surface, the agonist activates the target to produce a biological response. Agonists include hormones, neurotransmitters, antibodies, and antibody fragments.

Non-limiting examples of molecules that activate T cells include, but are not limited to, antibodies, such as whole antibodies and/or antibody fragments, NANOBODY® binding agents, AFFIMER® binding agents, and other molecular binding agents such as ligands and receptors.

The term “antibody” generally refers to an immunoglobulin (Ig) molecule consisting of four polypeptide chains, two heavy (H) and two light (L) chains, or functional fragments thereof that retain the epitope binding characteristics of the Ig molecule, including mutations. Refers to body, variant or derivative. Such fragment, mutant, variant or derivative antibody formats are known in the art. In one embodiment of a full-length antibody, each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain variable region (domain) is also designated as VDH in this disclosure. CH consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). CL consists of a single CL domain. The light chain variable region (domain) is also designated herein as VDL. VH and VL can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Generally, each VH and VL consists of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

In some embodiments, the immortalized cells express molecules that are antibody fragments. In some embodiments, the antibody fragment is selected from single chain variable fragment (scFv), F(ab')2 fragment, Fab fragment, Fab' fragment, and Fv fragment.

The term “fragment,” as used in conjunction with the term agonist or antibody, refers to a fragment of an agonist or antibody that retains the ability to specifically bind to an antigen. Examples of antibody fragments include (i) the Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) the F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of VH domain and CH1 domain; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of the antibody; (v) dAb fragment containing a single variable domain; and (vi) an isolated complementarity determining region (CDR). Additionally, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be generated using recombinant methods by pairing the VL and VH regions into a monovalent molecule, known as a single-chain Fv (scFv). ) can be linked by a synthetic linker, which allows them to be made as a single protein to form a protein. Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Other types of single chain antibodies, such as diabodies, are also included. Single chain antibodies also include "linear antibodies" which contain a pair of tandem Fv segments (VH-CH1-VH-CH1) that together with complementary light chain polypeptides form a pair of antigen binding regions.

The term “KD” refers to the dissociation equilibrium constant of a particular agonist-antigen interaction. Typically, the agonists described herein have a molecular ^weight of about 1 Binds to the target with a dissociation equilibrium constant (KD) higher than the KD of mOKT3 scFv, which is M or 1×10 ^-10 M. In some embodiments, an agonist described herein (e.g., a low affinity mOKT3 scFv variant) binds a target protein (e.g., CD3) with an affinity corresponding to a KD higher than 5×10 ^-10 M. do.

The term “koff” (sec-1) refers to the dissociation rate constant of a particular agonist-antigen interaction. This value is also referred to as the kd value.

The term “kon” (M-1Хsec-1) refers to the association rate constant of a specific agonist-antigen interaction.

The term “KD” (M) refers to the dissociation equilibrium constant of a particular agonist-antigen interaction.

The term “KA” (M ^-1 ) refers to the association equilibrium constant of a particular agonist-antigen interaction and is obtained by dividing kon by koff.

The phrase “anti-CD28 antibody” refers to an antibody or variant thereof, e.g., a monoclonal antibody, and includes human, humanized, chimeric or murine antibodies directed against the CD28 receptor on the T cell antigen receptor of mature T cells. do.

The phrase “anti-CD2 antibody” refers to an antibody or variant thereof, e.g., a monoclonal antibody, and includes human, humanized, chimeric or murine antibodies directed against the CD2 receptor at the T cell antigen receptor on mature T cells. do.

The phrase “anti-CD3 antibody” refers to an antibody or variant thereof, e.g., a monoclonal antibody, and includes human, humanized, chimeric or murine antibodies directed against the CD3 receptor on the T cell antigen receptor of mature T cells. (See, for example, International Publication No. WO2013186613A1, incorporated herein by reference). Anti-CD3 antibodies include OKT3, also known as muromonap. Anti-CD3 antibodies include the UCHT1 clone, also known as T3 and CD3c. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and vicilizumab.

Muromonab-CD3 light chain

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRADTAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTS TSPIVKSFNRNEC (SEQ ID NO: 17)

Muromonab-CD3 heavy chain

QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSS VTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18)

The term “OKT-3” refers to an anti-CD3 antibody produced by Miltenyi Biotech, Inc., San Diego, CA, USA, and/or a biosimilar or variant thereof (e.g., humanized, chimera or affinity mature variant). Hybridomas capable of producing OKT3 are available from the American Type Culture Collection and are assigned ATCC accession number CRL 8001. Hybridomas capable of producing OKT3 are available from the European Collection of Authenticated Cell Cultures (ECACC) and are assigned catalog number 86022706.

In some embodiments, the antibody fragment is an OKT3 antibody, such as an OKT3 antibody fragment. In some embodiments, the OKT3 antibody fragment is a membrane bound OKT3 (mOKT3) scFv fragment.

The transmembrane domain can be utilized to anchor mOKT3 scFv to the cell surface of immortalized cells. For example, the human CD8 transmembrane domain can be utilized to anchor mOKT3 scFv to the cell surface of immortalized cells. Also contemplated herein is the use of the CD8 transmembrane domain from another species, such as mouse (pKSQ366), or another transmembrane protein, such as CD14 or CD28, to anchor the mOKT3 scFv to the cell surface of immortalized cells. In some embodiments, the mOKT3 scFv is expressed tethered to the cell surface of an immortalized cell. In some embodiments, the human CD8 transmembrane domain is utilized to anchor the mOKT3 scFv to the cell surface of immortalized cells.

Many T cells will respond to very strong stimulation of mOKT3. The use of lower affinity T cell receptor binding allows for the isolation of subtle differences in T cell receptor signaling thresholds due to cell-to-cell variation and allows for more sensitive determination of quality between T cell therapy products prior to infusion into patients. . Low-affinity variants of the mOKT3 fragment can be created through site mutagenesis, which can more closely model the affinity of the recognized antigen and the natural T cell receptor. The published affinities of natural T cell receptors are in the μM KD range.

In some embodiments, low affinity mOKT3 scFv variants are used. In some embodiments, the molecule binds CD3 with a KD that is lower than the dissociation constant (KD) of the mOKT3 scFv, wherein the KD of the mOKT scFv is from about 1×10 ^-9 to about 1×10 ^-11 , e.g., about 5 ×10 ^-10 M. In some embodiments, the low affinity mOKT3 scFv variant comprises an amino acid sequence with an R55 mutation relative to the amino acid sequence of SEQ ID NO:2. In some embodiments, the low affinity mOKT3 scFv variant comprises an amino acid sequence with a Y57 mutation relative to the amino acid sequence of SEQ ID NO:2. In some embodiments, the low affinity mOKT3 scFv variant comprises an amino acid sequence with R55 and Y57 mutations relative to the amino acid sequence of SEQ ID NO:2. In some embodiments, the low affinity mOKT3 scFv variant comprises an amino acid sequence with the R55M and Y57A mutations relative to the amino acid sequence of SEQ ID NO:2. In some embodiments, the low affinity mOKT3 scFv variant comprises an amino acid sequence with the R55L and Y57T mutations relative to the amino acid sequence of SEQ ID NO:2.

In some embodiments, the low affinity mOKT3 scFv variant binds CD3 with a dissociation constant (KD) of about 1×10 ^-6 to about 5×10 ^-8 . For example, low-affinity mOKT3 scFv variants can bind CD3 with a KD of approximately 1×10 ^-6 , 1×10 ^-7 or 1×10 ^-8 . In some embodiments, the low affinity mOKT3 scFv variant binds CD3 with a KD of about 5×10 ⁻⁷ .

In some embodiments, the low affinity mOKT3 scFv variant has a KD that is at least 10-fold lower, at least 20-fold lower, at least 30-fold lower, at least 40-fold lower, at least 50-fold lower, at least 60-fold lower. lower, at least 70 times lower, at least 80 times lower, at least 90 times lower, at least 100 times lower, at least 110 times lower, at least 120 times lower, at least 130 times lower, at least 140 times lower, at least 150 times lower, at least 160 times lower, at least 170 times lower, at least 180 times lower, at least 190 times lower, at least 200 times lower, at least 210 times lower, at least 220 times lower, at least 230 times lower, at least 240 times lower, at least 250 times lower, at least 275 times lower, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, Binds to CD3 with a KD that is at least 1000-fold, at least 1100-fold, and at least 1200-fold lower. In some embodiments, the low affinity mOKT3 scFv variant binds CD3 with a KD that is at least 250-fold lower than the dissociation constant KD of the mOKT3 scFv. In some embodiments, the low affinity mOKT3 scFv variant binds CD3 with a KD that is at least 1000-fold lower than the dissociation constant KD of the OKT3 scFv.

In some embodiments, the molecule that activates the T cell is a membrane tethered molecule. Other molecules that activate T cells by binding to and clustering on the T cell receptor CD3, such as bacterial superantigens (e.g., SEB), phytohemagglutinin (PHA), or concanavalin A (ConA), can be used to activate T cells. It is considered to operate in a similar manner to mOKT3 scFv tethered to the cell surface. In some embodiments, the molecule that activates T cells is phytohemagglutinin (PHA). In some embodiments, the molecule that activates T cells is concanavalin A (ConA). Other monoclonal antibody scFv fragments that bind to the T cell receptor/CD3 component, such as anti-CD3 antibody clone BC3, are also contemplated to function in a similar manner to the mOKT3 scFv tethered to the cell surface of immortalized cells. In some embodiments, the molecule that activates T cells is anti-CD3 antibody clone BC3.

In some embodiments, the immortalized cells express Fc receptors. In this embodiment, antibodies that bind both Fc receptors and T cell antigens such as CD3, such as monoclonal anti-CD3 antibodies, can be used to assess the efficacy of TILs.

In another embodiment, the molecules that activate T cells are not expressed by the immortalized cells. Rather, the molecule may be a bispecific molecule capable of binding to both immortalized cells and T cells. Bispecific T cell engagers, such as blinatumomab, are a non-limiting example of such molecules that bind to CD19 expressed by immortalized cells and CD3 expressed by T cells.

Coculture conditions

In some aspects, the methods provided herein include co-culturing lymphocytes (e.g., TILs) and immortalized cells.

Immortalized cells can be cultured as two-dimensional monolayers of cells or as multilayered three-dimensional spheroids. Spheroids are self-assembling multicellular aggregates that form in environments that prevent attachment to flat surfaces.

Two-dimensional culture is an adherent culture in which cells grow as a monolayer, for example, in culture flasks, dishes, or multiwell plates with an adhesive surface. In comparison, three-dimensional spheroid culture is a non-adherent or low-adherence culture system in which cells are grown as multilayer spheroids, for example, in suspension culture on non-adherent plates, in concentrated media, or on gel-like materials or scaffolds. .

In some embodiments, immortalized cells in a three-dimensional system are cultured on ultra-low adhesion (ULA) surfaces. Ultra-low adhesion surfaces are surfaces containing substances that inhibit specific and non-specific immobilization, allowing cells to float and form three-dimensional spheroids. In some embodiments, the ultra-low adhesion surface includes a hydrophilic neutral charge coating. In some embodiments, the hydrophilic neutrally charged hydrogel coating is covalently attached to the surface. In some embodiments, the surface includes polystyrene. Accordingly, in some embodiments, the ultra-low adhesion surface is a polystyrene surface to which a hydrophilic neutral charged coating is covalently attached. As is known in the art, ultra-low adhesion surfaces are generally stable, non-cytotoxic, biologically inert, and non-degradable.

In some embodiments, immortalized cells in a three-dimensional system are cultured in ultra-low attachment multiwell plates (e.g., Corning®). In some embodiments, the ultra-low adhesion surface of a multiwell plate (e.g., a multiwell polystyrene plate) is a hydrophilic neutrally charged covalent hydrogel layer. A variety of multiwell formats are available, such as 6-well, 24-well or 96-well formats.

The culture conditions provided below can be used for two-dimensional or three-dimensional co-culture.

In some embodiments, the co-culture of immortalized cells includes 25,000 to 200,000 immortalized cells. For example, a co-culture of immortalized cells may contain 50,000 to 100,000 immortalized cells. In some embodiments, the co-culture of immortalized cells comprises 25000, 30000, 40000, 50000, 60000, 70000, 80000, 90000 or 10000 immortalized cells.

In some embodiments, the immortalized cells are grown in medium containing serum (e.g., fetal bovine serum (FBS)) and antibiotics (e.g., Pen/Strep), e.g., in a multiwell plate, e.g., a 96-well plate. Plated on (e.g., Dulbecco's modified Eagle's medium (DMEM)).

In some embodiments, the immortalized cells are cultured in medium (e.g., on a ULA surface) for about 48 hours to about 96 hours (e.g., about 48 hours, about 60 hours, about 72 hours, about 84 hours, or about 96 hours). or in adherent multiwell plates) to form three-dimensional spheroids prior to addition of TILs.

For example, to generate TILs, e.g., pre-REP TILs, prior to co-culture, in some embodiments, a tumor digest sample (e.g., a melanoma tumor digest sample) is thawed and incubated in the presence of IL-2. Plate in pre-REP medium (e.g., 1.5e6 cells/ml). In some embodiments, replating and feeding of IL-2 is repeated every 1 to 3 days until growth slows to less than 1.5-fold growth, for example, after 2 days.

The day before co-culture initiation, in some embodiments, pre-REP TILs are thawed and rested overnight in REP medium (e.g., RPMI, AIM-V, 5% human AB serum and IL-2).

In some embodiments, pan T cells are isolated from peripheral blood mononuclear cells (PBMC) and cultured with immortalized cells.

In some embodiments, co-culture is performed at a 2:1 effector (T cell) to target (immortalized cell) ratio (abbreviated as E:T). In some embodiments, co-culture is performed at a 1:1 E:T ratio. In some embodiments, co-culture is performed at a 1:2 E:T ratio. In some embodiments, co-culture is performed at a 3:1, 2:1, or 1:1 E:T ratio. In some embodiments, co-culture is performed at a 5:1 E:T ratio. In some embodiments, co-culture is performed at a 10:1 E:T ratio. In some embodiments, co-culture is performed at a 20:1 E:T ratio.

The co-culture period may vary. In some embodiments, the TIL and the immortalized cell are co-cultured for at least 2 hours, at least 3 hours, or at least 4 hours. For example, TILs and immortalized cells 2 to 72 hours, 2 to 48 hours, 2 to 36 hours, 2 to 24 hours, 2 to 12 hours, 4 to 72 hours, 4 to 48 hours, 4 to 36 hours, 4 to 24 hours, 4 to 12 hours, 12 to 72 hours, 8 to 72 hours, 8 to 48 hours, 8 to 36 hours, 8 to 24 hours, 8 to 12 hours, 12 to 72 hours, 12 to 48 hours, 12 Co-culture may be performed for 36 hours, 12 to 24 hours, 24 to 72 hours, 24 to 48 hours, or 24 to 36 hours. In some embodiments, TILs and immortalized cells are co-cultured for about 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 72 hours. In some embodiments, the TIL and the immortalized cells are cultured for at least 12 hours, at least 24 hours, at least 48 hours, at least 36 hours, or at least 72 hours.

In some embodiments, co-culture of TILs with immortalized cells produces interleukin-2 (IL-2), IL-15, or a combination thereof, e.g., at about 5,000 U/ml to about 8,000 U/ml (e.g. , 5,000U/ml, 5,500U/ml, 6,000U/ml, 6,500U/ml, 7,000U/ml, 7,500U/ml or 8,000U/ml).

IL-2 is an interleukin, a type of cytokine signaling molecule in the immune system. It is a 15.5 to 16 kDa protein that regulates the activity of white blood cells (leukocytes, often lymphocytes) responsible for immunity. IL-2 is part of the body's natural response to microbial infection. IL-2 mediates its effects by binding to the IL-2 receptor expressed on lymphocytes. The main sources of IL-2 are activated CD4+ T cells and activated CD8+ T cells.

IL-2 plays an essential role in key functions of the immune system, tolerance and immunity, primarily through direct effects on T cells. In the thymus, where T cells mature, they prevent autoimmune diseases by promoting the differentiation of certain immature T cells into regulatory T cells, which suppress other T cells that would otherwise be primed to attack normal healthy cells in the body. do. IL-2 enhances activation-induced cell death (AICD). IL-2 also promotes the differentiation of T cells into effector T cells and memory T cells when initial T cells are stimulated by antigen, helping the body fight infection. Together with other polarizing cytokines, IL-2 stimulates the differentiation of naïve CD4+ T cells into Th1 and Th2 lymphocytes, whereas it impedes differentiation into Th17 and follicular Th lymphocytes. Its expression and secretion are tightly regulated and function as part of transient positive and negative feedback loops that potentiate and dampen immune responses. Through its role in the development of T cell immunological memory, which depends on the expansion of the number and function of antigen-selected T cell clones, it serves to sustain cell-mediated immunity.

IL-15 is a 14 to 15 kDa glycoprotein encoded by a 34 kb region on chromosome 4q31 in humans. IL-15 is a cytokine with structural similarity to IL-2. Like IL-2, IL-15 binds to and transmits signals through a complex consisting of the IL-2/IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 is constitutively expressed in numerous cell types and tissues, including monocytes, macrophages, dendritic cells (DCs), keratinocytes, fibroblasts, myocytes, and neurons. As a pleiotropic cytokine, it plays an important role in innate and adaptive immunity. IL-15 regulates the activation and proliferation of T cells and natural killer (NK) cells. Survival signals that maintain memory T cells in the absence of antigen are provided by IL-15. These cytokines are also involved in NK cell development.

Evaluation of lymphocyte antitumor efficacy

After co-culture of immortalized cells with lymphocytes (e.g., TILs), the lymphocyte anti-tumor potency can be determined by, for example, degranulation of the immortalized cells over a period of time (e.g., CD107a expression), cytokine production (e.g. , IFN-γ) and/or cell death and/or viability can be measured. For example, the period for evaluating effectiveness may be 12 to 72 hours. In some embodiments, lymphocyte (e.g., TIL) anti-tumor potency is assessed for 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, 24 to 72 hours, 24 to 48 hours, or 24 to 36 hours. . In some embodiments, lymphocyte (e.g., TIL) antitumor efficacy is assessed over 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours. In some embodiments, lymphocyte (e.g., TIL) anti-tumor efficacy is assessed for at least 12 hours, at least 24 hours, at least 48 hours, at least 36 hours, or at least 72 hours.

Methods for measuring degranulation include, but are not limited to, cell surface staining for lysosomal associated membrane glycoproteins (LAMPs) such as CD107a or CD107b using flow cytometry readout. LAMP is found in the lipid bilayer of cytolytic granules, and when released to mediate death by TILs, it fuses to the TIL surface and serves as a marker for degranulation and direct cytotoxicity.

Methods for measuring cytokine products include ELISA, Luminex or MSD assays of cell culture supernatants after co-culture of TILs with the mOKT3-A375 cell line, or qRT-PCR analysis of cytokine transcripts in TILs after co-culture of mOTK3-A375 cell line, but these is not limited to

Methods for measuring cell death and/or viability to assess potency include real-time cell viability assay, ATP cell viability assay, live cell protease viability assay, tetrazolium reduced cell viability assay, and resazurin reduced cell viability assay. , dead cell protease release cytotoxicity assay, lactate dehydrogenase release cytotoxicity assay, and DNA dye cytotoxicity assay. Other assays known in the art for measuring cell health, cell death, and/or cell viability are also contemplated herein. Non-limiting examples of such assays are as follows.

Cell viability assays use various markers as indicators of metabolically active (living) cells. Examples of commonly used markers include measuring ATP levels, measuring substrate reducing capacity, and detecting enzyme/protease activity specific to living cells.

The RealTime-Glo™ MT Cell Viability Assay (Promega, catalog number G9711) measures cell viability in real time. In this assay, engineered luciferase and prosubstrate (not a substrate of luciferase) are added directly to the culture medium. The precursor substrate can penetrate the cell membrane and enter the cell. However, only viable cells with active metabolism can reduce the precursor substrate to a substrate for luciferase. The substrate then exits the cell where it is used by luciferase in the detection reagent to generate a luminescent signal. The same well can be measured repeatedly over 3 days. The main advantage of this method is that dose response can be determined using fewer plates and cells through simple kinetic monitoring. Additionally, because this method does not require cell lysis, the same cells can be used for additional cell-based assays or downstream applications.

Because only viable cells can synthesize ATP, ATP can be used to measure cell viability. ATP can be measured using the CellTiter-Glo® luminescent cell viability assay (Promega, catalog number G7570) using reagents containing detergent, stabilized luciferase, and luciferin substrate. Detergent lyses viable cells, releasing ATP into the medium. In the presence of ATP, luciferase uses luciferin to produce luminescence, which can be detected within 10 minutes using a luminometer. CellTiter-Glo® 2.0 assay (Promega, catalog number G9241) is available as a single solution that reduces reagent preparation time and provides room temperature storage for easy implementation. This ATP assay does not require long incubation times to convert the substrate to a colored product. Additionally, it has excellent sensitivity and wide linearity, making it well compatible with high-throughput applications where small cell numbers are used. It is also less likely to produce artifacts than other methods.

Live cell protease activity disappears rapidly after apoptosis, so this is a useful marker of viable cells. The CellTiter-Fluor™ Cell Viability Assay (Promega, catalog number G6080) allows measurement of live cell protease activity using a cell-permeable fluorogenic protease substrate (GF-AFC). The substrate enters live cells, where it is cleaved by live cell proteases, producing a fluorescent signal proportional to the number of viable cells. The incubation time for this method is 0.5 to 1 hour, which is shorter than the tetrazolium assay (1 to 4 hours). Because this method does not lyse cells, it allows multiplexing with many other assays in the same sample well, including bioluminescent reporter cell-based assays.

Tetrazolium compounds used to detect viable cells fall into two basic categories:

Positively charged compounds (MTT) that readily penetrate viable cells:

Viable cells with active metabolism can convert MTT to a purple formazan product. Therefore, color formation may be a useful marker of viable cells. The CellTiter 96 ^® non-radioactive cell proliferation assay (MTT) (Promega, catalog number G4000) uses this chemistry. However, the incubation time for this method is long (usually 4 hours). Additionally, because the formazan product is insoluble, a solubilizing reagent must be added before recording absorbance readings.

Negatively charged compounds that do not penetrate cells (MTS, XTT, WST-1):

When using the CellTiter 96 ^® Aqueous 1 Solution Cell Proliferation Assay (MTS) (Promega, catalog number G3582), a negatively charged compound must be combined with an intermediate electron coupling reagent, which enters the cell, is reduced, and then exits the cell to form tetrazolium. It can be converted to a soluble formazan product. Incubation time for this method is 1 to 4 hours. Since the produced formazan is soluble, there is no need to add a solubilizer, which is more convenient.

Resazurin is a dark blue, cell-permeable indicator dye with little intrinsic fluorescence. The CellTiter-Blue ^® cell viability assay (Promega, catalog number G8080) uses resazurin to measure cell viability. Only living cells with active metabolism can reduce resazurin to pink fluorescent resorufin. After 1 to 4 hours of incubation, quantify the signal using a microplate spectrophotometer or fluorometer. This method is relatively cheaper and more sensitive than the tetrazolium assay. However, fluorescence from the compound being tested can interfere with the resorufin readout.

A drawback of all tetrazolium or resazurin reduction assays is that they depend on the accumulation of colored or fluorescent products over time. Because the signal increases gradually over time, no decrease in cell viability can be detected during these long incubations.

When cells die and lose the integrity of their membranes, apoptotic cell proteases are released. Apoptotic cell protease activity can then be measured using a luminescent substrate (CytoTox-Glo™ cytotoxicity assay, Promega, catalog number G9290) or a fluorescent substrate (CytoTox-Fluor™ cytotoxicity assay, Promega, catalog number G9260). Because the matrices are not cell permeable, essentially no signals from intact viable cells are generated from these matrices. Additionally, the black is insoluble, so it can be multiplexed with other compatible black chemistries.

Dead cells that have lost their membrane integrity release lactate dehydrogenase (LDH), which catalyzes the conversion of lactate to pyruvate and concurrent production of NADH. Released LDH activity can be measured by providing excess substrate (lactate and NAD+) to produce NADH. This NADH can be measured using a variety of assay chemistries.

1. LDH-Glo™ cytotoxicity assay: In the LDH-Glo™ cytotoxicity assay (catalog number J2380), reductase uses NADH and a reductase substrate (proluciferin) to produce luciferin. Luciferin is measured using a proprietary luciferase and the optical signal is proportional to the amount of LDH measured with a luminometer.

2. CytoTox-ONE™ Homogenous Membrane Integrity Assay: CytoTox-ONE™ Homogenous Membrane Integrity Assay (Promega, Cat. No. G7890): Conversion of resazurin to the fluorescent resorufin product measured using a fluorometer.

3. CytoTox 96® Non-radioactive Cytotoxicity Assay: The CytoTox 96® Non-radioactive Cytotoxicity Assay (Promega, catalog number G1780) detects the conversion of the tetrazolium salt (INT) to the red formazan product as measured by color absorbance.

Some DNA-binding dyes are excluded from living cells but can enter permeable dead cells and stain their DNA. Conventional dyes such as trypan blue often require manual counting of stained cells using a hemocytometer, which is labor intensive and cannot be easily scaled up. Another disadvantage of conventional dyes is that they can be toxic to cells and can only be used for endpoint measurements.

Modern dyes, such as CellTox™ Green Dye, produce a fluorescent signal when bound to DNA, which is easily measured using a fluorometer. It can be diluted in culture medium and delivered directly to cells at seeding or when treated with test compounds, allowing real-time kinetic measurements. The CellTox™ green cytotoxicity assay (Promega, catalog number G8741) is non-toxic, highly photostable, and can be easily scaled up.

All references, patents, and patent applications disclosed herein are each incorporated by reference with respect to the subject matter cited, and in some cases may encompass the entire document.

As used in this specification and claims, the singular expressions “a,” “a,” and “an” should be understood to mean “at least one,” unless clearly indicated otherwise.

Additionally, unless clearly indicated to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited. You must understand that it is not.

In the foregoing specification as well as in the claims, all transitions such as "comprising", "comprising", "carrying", "having", "containing", "comprising", "having", "consisting of", etc. The phrase should be understood to mean open-ended, including but not limited to. As specified in Section 2111.03 of the United States Patent and Trademark Office Patent Examination Procedure Manual, only the transition phrases “consisting of” and “consisting essentially of” are closed or semi-closed transition phrases, respectively.

The terms “approximately” and “substantially” preceding a value mean ±10% of the quoted value.

Where a range of values is provided, each value between and including the upper and lower limits of the range is specifically contemplated and described herein.

Example

Example 1: Generation of the universal T cell agonist protein MOKT3 and its lower affinity variants

The universal human T cell agonist (referred to herein as membrane-OKT3 (mOKT3) or pKSQ367) was generated by fusion of the following amino acid sequences:

Signal peptide from mouse IgG heavy chain: MERHWIFLLLLSVTAGVHS (SEQ ID NO: 1);

scFv from mouse monoclonal anti-CD3e antibody clone OKT3:

QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDT SKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEIN (SEQ ID NO: 2); and

Transmembrane domain from human CD8:

SHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV (SEQ ID NO: 12).

A codon-optimized cDNA encoding the mOKT3 protein was cloned containing the EF1A promoter followed by the T2A self-cleaving peptide (EGRGSLLTCGDVEENPGP (SEQ ID NO: 15)) and the blasticidin resistance gene (MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTGVNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGIKAIVKDSDGQPTAV Lentiviral vector containing GIRELLPSGYVWEG* (SEQ ID NO: 16)) Cloned into plasmid. The final construct with OKT3-CD8 protein expressed by the EF1A promoter is also referred to herein as pKSQ367.

Lower affinity variants of the mOKT3 protein were constructed by site-directed mutagenesis of the pKSQ367 lentiviral construct. Positions R55 and Y57 of scFv were described by Chen et al . Mutations were made to R55M and Y57A (known as mOKT3ma/pKSQ397), or R55L and Y57T (known as mOKT3lt/pKSQ398), respectively, as described in doi.org/10.3389/fimmu.2017.00793. The published affinities of these scFv variants are either 250-fold lower (OKT3ma) or 1000-fold lower (OKT3lt) than the parent OKT3 clone (KD, published affinities of 5×10 ^-10 M, Law et al, Reinhertz et al.). Low affinity variants of mOKT3 more closely model the affinity of the natural T cell receptor (TCR) and the recognized antigen (public affinities in the μM KD range). While many T cells respond to very strong stimulation of mOKT3, lower affinity TCR binding allows for isolating subtle differences in cell-to-cell TCR signaling thresholds and further ensures quality between T cell therapy products prior to infusion into patients. Allows you to make sensitive decisions.

Lentiviral constructs were packaged by co-transfecting pCMV-VSV-G and psPax2 lentiviral packaging plasmids into 293T cells. Viral supernatant was transferred to A375 cells and successfully transduced cells carrying the mOKT3 construct were selected by switching the medium to blasticidin-containing medium 48 hours after transduction. Successfully transduced and selected A375 cells will be referred to as A375-pKSQ367 (also known as A375-mOKT3).

Example 2: Generation of mOKT3-A375 cells and confirmation that they activate T cells.

50,000 or 100,000 A375-pKSQ367 cells were plated per well in 96-well plates in Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and antibiotics. Pan T cells were isolated from peripheral blood mononuclear cells (PBMC) and cocultured with previously plated A375-pKSQ367. Cocultures were performed at a 2:1 effector to target ratio (abbreviated as E:T). Media was removed from previously plated A375-pKSQ367, and 100,000 or 200,000 T cells in hematopoietic serum-free culture medium containing IL-2 were added to wells containing 50,000 or 100,000 A375-pKSQ367, respectively. . The supernatant was removed from the plate and the cells were stained with anti-CD3 and anti-CD69 antibodies. Flow cytometry was performed and CD69 expression was measured. CD69 activation was observed in all transduced samples ( Figure 1 ).

To confirm the surface expression of mOKT3, A375-pKSQ367 cells were incubated with goat serum in cell staining buffer, then cells were washed with cell staining buffer and then incubated with 1:100 goat-anti-mouse antibody. Cells were washed and flow cytometry was performed to assess expression. OKT3 expression was observed in 99.2% of transduced cells ( Figure 2 ).

Killing of mOKT3-A375 cells by tumor infiltrating lymphocytes (TILs)

To generate pre-REP TIL, melanoma tumor digestion samples were thawed and incubated with heat-inactivated human AB serum, Pen/Step, HEPES buffer, Glutamax, beta-mercaptoethanol, gentamicin, IL-2, and DNase. were plated at 1.5e6 cells/ml in pre-REP medium containing (added only to D277 and D3291 cultures). IL-2 was added to the cells later. Count cells and repopulate at 1e6 cells/ml in pre-REP medium with IL-2 (D277 and D3291) or 1:1 pre-REP TIL medium: hematopoietic serum-free culture medium and IL-2 (D5746). ting. Replating and feeding of IL-2 was repeated after 2 days until growth slowed to less than 1.5-fold growth. D277, D3291, and D5746 were frozen after the incubation period.

The day before the start of co-culture, pre-REP TILs were thawed from three donors and allowed to stand overnight in REP medium containing IL-2. 6,000 A375-pKSQ367 were plated in 96-well plates the day before the start of co-culture in REP medium containing IL-2 and caspase 3/7 dye. The next day, the medium was removed from the 96-well plate, and TILs were added to A375-pKSQ367 plated the previous day at 10:1, 3:1, 1:1, or 0.3:1 E:T. Plates were imaged by IncuCyte every 2 hours for 3 days. TIL recognition and killing of A375-pKSQ367 cells was observed in all E:Ts tested ( Figures 3A to 3C ).

Examples of Alternative OKT3 Membrane Anchors:

The purpose of this experiment was to show that various signal peptides and membrane anchors can be used to express mOTK3 on the cell surface and that mOKT3 can be expressed in other cell types besides A375 cells. pKSQ328, pKSQ377 containing an OKT3 scFv fused to various signal peptides such as mouse IgG, human CSF2R and human CD5, and membrane anchor proteins including mouse CD8a, human CD8a, human CD28 and human CD14 in 2 million K562 cells. , lentiviruses encoding pKSQ378, pKSQ368, and pKSQ369 constructs were transduced. Forty-eight hours after transduction, transduced K562 cells were selected for blasticidin resistance markers, if present. At day 8 after transduction, the K562 stable cell line was plated at 100,000 cells/well in the presence of 100,00 pan T cells isolated from donor PBMCs in IL-2 containing medium. 24 hours after plating, cells were stained with anti-CD8 and anti-CD69 antibodies. Flow cytometry was performed and CD8+ T cells were quantified for surface expression of CD69 (a marker of T cell activation) compared to unstimulated controls. We observed that various combinations of signal peptide and membrane anchor used to construct the mOKT3 construct induced activation of T cells (Figure 4).

Example 3: Characterization: expression of mOKT3, co-culture with pan T cells and cd69 induction

A375 parental cells (containing NucLightRed, a red fluorescent nuclear reporter) were transduced with pKSQ367, pKSQ396, pKSQ397, or pKSQ398. The 'low affinity' constructs pKSQ396, pKSQ397 and pKSQ398 were transduced with three different volumes of virus to ensure transduction.

Several assays were performed to evaluate the efficacy of the transduction and constructs.

1. mOKT3 expression on the cell surface.

2. Activation of pan T cells through expression of CD69 after co-culture with A375 transduced target cells.

3. Reduced growth (indicative of recognition by TILs) of A375 transduced cells after co-culture with patient-derived melanoma TILs.

A375 cells (both A375 parental and transgenic lines) were resuspended in cell culture medium at 1.5e6/ml and then plated in 96 well V-bottom plates at 0.75e5/50 μl/well and incubated overnight. The next day, plated cells were blocked with goat serum in cell staining buffer and incubated in the dark. Cells were then stained and incubated with goat anti-mouse IgG2a polyclonal Alexa647 (1:100) in cell staining buffer. After washing, cells were resuspended in cell staining buffer and acquired on a BD Fortessa ( Figures 5-7 ).

A375 cells (both A375 parental and transgenic lines) were resuspended in cell culture medium at 1.5e6/ml and then plated in 96 well V-bottom plates at 0.75e5/50 μl/well and incubated overnight. next day; PBMCs (donor #148192) were thawed and pan T cells were isolated according to the manufacturer's protocol. Isolated Pan T cells were counted and resuspended at 1.5e6/ml. 0.75e5 isolated pan T cells were added to the plated A375 target cells (50 μl per well) and incubated overnight. Plated cells were washed with cell staining buffer, stained against anti-CD69 Ab BV785 in cell staining buffer, and incubated. After washing, cells were resuspended in cell staining buffer and acquired on a BD Fortessa ( Figures 8 to 11 ).

Example 4: Two-dimensional A375-pKSQ367 co-culture model

A375 cells (both A375 parental and transgenic lines) were resuspended at 1.2e5/ml in cell culture medium and then plated at 6e3/50 μl/well in 96-well flat bottom plates and incubated overnight. next day; 6e3/50 μl/well of each TIL condition in REP medium (AIMV, RPMI, Human AB Serum) was added onto the plated A375 target cells. One REP medium containing IL-2 was added to each coculture well. Coculture plates were incubated at room temperature before transferring to IncuCyte. Coculture plates were incubated in IncuCyte for an additional hour before starting image acquisition. Images were acquired every 2 hours for a total of 72 hours (Read Phase & Red at 10x objective). IncuCyte images were quantified for the total area of red fluorescence detected in each well and then normalized back to 0 hours to confirm changes in target cell expansion/decrease over time ( FIGS. 12 to 14 ).

In Figures 13 and 14 , A375 pKSQ367 and A375 pKSQ398 cells were cultured as a monolayer, respectively. Unedited and gene-edited TILs were added to the culture, and TIL cytotoxicity was measured as changes in tumor cell area through imaging. For A375 pKSQ367 as a tumor target, neither SOCS1 nor CBLB gene edited TILs showed better tumor control than unedited TILs ( Figure 13 ); For A375 pKSQ398 as a tumor target, all TILs, whether edited or unedited, were unable to control tumor growth ( Figure 14 ). Therefore, in an environment where tumor targets are cultured as a monolayer, the efficacy of unedited and edited TILs cannot be differentiated.

Example 5: 3D A375-pKSQ367 spheroid co-culture model

Since 3D tumor spheroids can provide more challenging targets and complex microenvironments than monolayers of target cells, we explored the use of A375-pKSQ367 spheroids as target cells for coculture assays. This allows us to identify subtle differences in efficacy between edited and unedited TILs.

Both high-affinity (pKSQ367) and low-affinity (pKSQ398) A375 lines generated from a single clone were used in spheroid-based cocultures.

A375-pKSQ367 high-affinity and low-affinity clones were maintained in DMEM supplemented with heat-inactivated FBS and antibiotics. A375-pKSQ367 high-affinity single cell clone 20 was used in the high-affinity spheroid co-culture assay, and A375-pKSQ367 low-affinity single cell clone 6 was used in the low-affinity spheroid co-culture assay.

For A375-pKSQ367 spheroid co-culture, the general protocol was followed:

제-4일: A375-pKSQ367을 FBS 및 항생제가 보충된 RPMI에서 계대배양하고 5,000 내지 10,000개의 생세포/웰의 밀도로 플레이팅하였다. 플레이트를 37℃ 5%CO₂ 인큐베이터 내부의 Incucyte 영상화 시스템으로 옮기고 스페로이드 형성을 촉진하기 위해 인큐베이션시켰다. 몇 시간마다 한 번씩 위상 및 적색 채널에서 4X 배율로 영상을 촬영하였다.

Day-4: A375-pKSQ367 was subcultured in RPMI supplemented with FBS and antibiotics and plated at a density of 5,000-10,000 viable cells/well. The plate was transferred to the Incucyte imaging system inside a 37°C 5%CO ₂ incubator and incubated to promote spheroid formation. Images were taken at 4X magnification in the phase and red channels once every few hours.

Day - 1: Cryopreserved TIL from 4 donors (Donor 1: D4267, NSCLC; Donor 2: D4397, NSCLC; Donor 3: D6164, melanoma; Donor 4: D6481, melanoma) were thawed, washed, and Counted and reproduced at 3e^6 viable cells per ml in TIL medium (RPMI 1640, AIM V, supplemented with human AB serum). IL-2 was added. Cells were placed in a flask of appropriate size and left overnight in an incubator at 37°C and 5% CO ₂ .

Day 0: TILs were filtered through a cell strainer into appropriately sized conical tubes, centrifuged, and counted. Plates containing A375-pKSQ367 spheroids were removed from Incucyte. TILs were added to A375-pKSQ367 spheroids at an effector cell to target cell (E:T) ratio of 10:1 to 0.625:1. E:T ratios were calculated based on day 0 live TIL (effector) counts and A375-pKSQ367 (target) initial seeding density. Some wells had no TIL added and served as growth controls. The plate was transferred to the Incucyte imaging system inside a 37°C 5%CO ₂ incubator and imaging continued as on day -4.

Day 6: Imaging was discontinued and analysis was performed by visually evaluating the images collected by Incucyte.

In this case, the image was converted to black and white and analysis was performed by comparing the area of spheroids (black) remaining in the well at a specific time point. This data was also analyzed by evaluating the fluorescence intensity of the spheroids. The decrease in fluorescence level was used as an indicator of spheroid death. This assay can also be designed to assess target cell apoptosis by reading chromium release or caspase 3 expression.

The morphology of A375-pKSQ367 spheroids was evaluated in wells without TIL added. Differences between high-affinity and low-affinity spheroid morphologies were observed. Specifically, at the same plating density (10,000 cells/well), the high-affinity strain (denoted as “A375-pKSQ367” in the figure) is significantly “larger” than the low-affinity strain (denoted as “A375-OKT3lt” in the figure). /"Loose" spheroids were formed. Morphological differences were observed during the spheroid co-culture period (4 to 10 days after A375-OKT3 seeding). It is possible that morphological differences may exist even between clones of the same lineage, and these morphological differences may provide another opportunity to uncover subtle differences between T cells ( Fig. 15 ).

The cytotoxicity of TILs on A375-pKSQ367 spheroids was assessed by comparing the effect of increased E:T ratio at the same time points. Coculture of A375-pKSQ367 spheroids with TILs resulted in dose-dependent killing by no-EP and SOCS1-edited TILs, both high-affinity (indicated as “A375-OKT3” in the figure) (donors 1 and 2) and low-affinity (indicated as “A375-OKT3” in the figure). A375-OKT3lt") (donors 1, 2, 3, and 4) were observed for both spheroids ( Figure 16 ).

The difference in the cytotoxic kinetics of TILs against A375-pKSQ367 high-affinity (indicated as “A375-pKSQ367” in the figure) and low-affinity (indicated as “A375-pKSQ367A375-pKSQ398” in the figure) spheroids is due to the difference in the cytotoxicity of TILs remaining at the same time point. The size of the igneous spheroid and the low-affinity spheroid were compared and evaluated. The present inventors observed that high-affinity spheroids were killed faster than low-affinity spheroids by donors 1 and 2 ( Figure 17 ).

In Figure 17 , where A375 pKSQ367(A375-OKT3) tumor cells were grown as 3D tumor spheroids for 4 days prior to TIL addition, SOCS1 edited TILs displayed a greater ability to kill tumors than the unedited control (no EP). , at a 1.25:1 ratio, tumors were completely eliminated by SOCS1-edited TILs, whereas residual tumor was recognized in wells with unedited controls. Enhanced efficacy of SOCS1-edited TILs was observed when A375 pKSQ398(A375-OKT3lt) cells were grown as 3D tumor spheroids, but differences were observed at higher E:T ratios (5:1).

We assessed whether SOCS1-edited TILs induced increased killing of high-affinity A375-pKSQ367 spheroids at the same E:T. In donor 2, increased cytotoxicity against high-affinity A375-pKSQ367 spheroids was observed by SOCS1-edited TILs. These images were captured after 5 days of co-culture, demonstrating that A375-pKSQ367 can distinguish TILs of different antitumor potency ( Figure 18 ).

We assessed whether SOCS1-edited TILs induced increased killing of low-affinity A375-pKSQ367 spheroids. For donors 2, 3 and 4, we observed increased potency against A375-pKSQ367 low affinity spheroids from SOCS1 edited TILs. These results demonstrate that A375-pKSQ367 low affinity spheroids increase the sensitivity to distinguish TIL populations of different potency ( Figure 19 ).

Whether SOCS1-edited TILs increased the killing of low-affinity A375-pKSQ367 spheroids was assessed by spheroid fluorescence. A375-OKT3lt cells were plated in 96-well ultra-low attachment plates and cultured for 4 days to allow spheroid formation. On day 4, SOCS1 edited TILs or control (e.g., unedited) TILs were thawed and added to the spheroid cultures at various effector:target (E:T) ratios. Cytotoxicity of spheroids by SOCS1 edited TILs or control (e.g., unedited) TILs was monitored by InCucyte according to changes in spheroid fluorescence levels. SOCS1-edited TILs showed increased killing of low-affinity A375-pKSQ367 spheroids with increasing E:T ratio ( Figure 20 ).

Example 6: Use of the A375-pKSQ367 model to evaluate TIL potency based on IFN-γ and/or IL-6 cytokine release

TIL potency may also be measured in the supernatant based on cytotoxicity and/or cytokine release. Low-affinity A375-pKSQ367 cell three-dimensional (3D) spheroids were used as target cells for coculture assays to evaluate cytotoxicity and effector cytokine release. High-(pKSQ367) lines generated from a single clone can also be used as described below.

A375-pKSQ367 high-affinity and low-affinity clones were maintained in DMEM supplemented with heat-inactivated FBS and antibiotics for single cell suspension and 3D spheroid coculture analysis.

For A375-pKSQ367 3D spheroid co-culture a similar protocol was followed:

Day -4 (for 3D spheroid co-cultures only): A375-pKSQ367 was subcultured in RPMI supplemented with FBS and antibiotics and plated at a density of 5,000-10,000 viable cells/well. The plate was transferred to the Incucyte imaging system inside a 37°C 5%CO ₂ incubator and incubated to promote spheroid formation. Images are taken at 4X magnification in the phase and red channels once every few hours.

Day-1: Cryopreserved TIL were thawed, washed once, counted, and seeded in TIL medium (1:1 mixture of RPMI 1640 and AIM V supplemented with human AB serum) at 3e6 viable cells per ml. It was resuspended. Add IL-2. Cells were placed in a flask of appropriate size and left overnight in an incubator at 37°C and 5% CO ₂ . For single cell suspension assays, A375-pKSQ367 cells were plated at a density of 5,000 to 10,000 viable cells/well. The plate was transferred inside a 37°C 5%CO ₂ incubator overnight before adding TIL.

Day 0: TILs were filtered through a cell strainer into appropriately sized conical tubes, centrifuged, and counted. Plates containing A375-pKSQ367 spheroids and/or A375-pKSQ367 single cell suspension were removed from the incubator. TILs were added at an effector cell to target cell (E:T) ratio of 20:1 to 0.625:1. E:T ratios were calculated based on day 0 live TIL (effector) counts and A375-pKSQ367 (target) initial seeding density. Some wells had no TIL added and served as growth controls. Plates were transferred to a 37°C 5%CO ₂ incubator or an Incucyte imaging system inside a 37°C 5%CO ₂ incubator as appropriate for the assay and imaging continued.

3D Spheroid Assay: Imaging was stopped between the first and second days after addition of TIL and analysis was performed by visually evaluating the images collected in Incucyte. The cytotoxicity of TILs on A375-pKSQ367 spheroids was assessed by comparing the effect of increased E:T ratios at this time point with supernatants collected simultaneously. Production of IFNγ and/or IL-6 and the presence of other cytokines were detected directly in the supernatant. As an alternative approach to assess TIL cytotoxicity, the level of lactate dehydrogenase, which is rapidly released into the supernatant upon target cell lysis, was also measured in the supernatant using the LDH-Glo ^™ cytotoxicity assay (Promega).

TILs with increased potency produce greater amounts of IFNγ and IL-6 cytokine production as assessed by ELISA, MSD, or Luminex assays in single cell suspension or spheroid co-culture assays and/or are produced by lactate dehydrogenase in the supernatant. It was predicted to exhibit greater cytotoxicity as reflected in increased measurements of genease.

We assessed whether SOCS1-edited TILs grown in co-culture with low-affinity A375-pKSQ367 spheroids increased the potency of IFNγ and/or IL-6. Increased E:T ratio of SOCS1 edited TILs cocultured with low-affinity A375-pKSQ367 spheroids indicated increased secretion of both IFNγ ( Figure 21 ) and IL-6 ( Figure 22 ). SOCS1 edited TILs produced higher levels of both cytokines across all E:T ratios.

Supernatants of cocultures of SOCS1-edited TILs and low-affinity A375-pKSQ367 cocultures also showed higher levels of lactate dehydrogenase compared to cocultures with control TILs, which was consistent with higher amounts of A375-KSQ367 cell death. and enhanced cytotoxicity leading to lactate dehydrogenase release ( Figures 23 and 24 )

Claims (111)

As a method for evaluating the potency of tumor infiltrating lymphocytes (TIL),
co-culturing a TIL and an immortalized cell, wherein the immortalized cell comprises a molecule that activates a T cell; and
Steps to evaluate the effectiveness of the TIL
Method, including. The method of claim 1 , wherein the molecule binds a T cell antigen. The method of claim 2, wherein the TIL expresses the T cell antigen. The method of claim 2 or 3, wherein the T cell antigen is a CD3 antigen. 5. The method of any one of claims 1 to 4, wherein the immortalized cell expresses the molecule. 6. The method according to any one of claims 1 to 5, wherein the molecule is an antibody or antibody fragment. 7. The method of claim 6, wherein the antibody fragment is selected from single chain variable fragment (scFv), F(ab')2 fragment, Fab fragment, Fab' fragment and Fv fragment. The method of claim 7, wherein the antibody fragment is scFv. The method of any one of claims 6 to 8, wherein the antibody or antibody fragment is an OKT3 antibody or OKT3 antibody fragment, respectively. The method of claim 9, wherein the OKT3 antibody fragment is membrane bound OKT3 (mOKT3) scFv. The method of claim 10, wherein the mOKT3 scFv is a low affinity mOKT3 scFv variant. 12. The method of any one of claims 4 to 11, wherein the molecule binds CD3 with a KD that is higher than the dissociation constant (KD) of the mOKT3 scFv, wherein the KD of the mOKT3 scFv is about 5×10 ^-10 M. method. The method of claim 11 or 12, wherein the low affinity mOKT3 scFv variant comprises an amino acid sequence with R55 and Y57 mutations compared to the amino acid sequence of SEQ ID NO:2. The method of claim 13, wherein the low-affinity mOKT3 scFv variant comprises an amino acid sequence with R55M and Y57A mutations compared to the amino acid sequence of SEQ ID NO:2. 15. The method of claim 14, wherein the low affinity mOKT3 scFv variant binds to CD3 with a KD that is at least 250 times higher than the dissociation constant KD of mOKT3 scFv. The method of claim 13, wherein the low-affinity mOKT3 scFv variant comprises an amino acid sequence having R55L and Y57T compared to the amino acid sequence of SEQ ID NO: 2. 17. The method of claim 16, wherein the low affinity mOKT3 scFv variant binds to CD3 with a KD that is at least 1000 times higher than the dissociation constant KD of OKT3 scFv. 6. The method according to any one of claims 1 to 5, wherein the molecule is a bacterial superantigen, optionally Staphylococcal enterotoxin B (SEB), phytohaemagglutinin (PHA) and Concana A method selected from valine A (concanavalin: ConA). 19. The method of any one of claims 1 to 18, wherein the molecule is a membrane-tethered molecule. 20. The method of any one of claims 1-19, wherein the TIL is an engineered TIL (eTIL). 21. The method of claim 20, wherein the eTIL is an edited eTIL. 22. The method of claim 21, wherein the edited eTIL comprises a genomic modification. 23. The method of any one of claims 1 to 22, wherein the immortalized cells comprise a genetic population of immortalized cells. 24. The method of any one of claims 1 to 23, wherein the immortalized cells are immortalized human cells. 25. The method of any one of claims 1-24, wherein the immortalized cells are engineered cancer cells. 26. The method of any one of claims 1-25, wherein the engineered cancer cells are selected from engineered melanoma cells, engineered colorectal cancer cells, engineered cholangiocarcinoma cells, and engineered breast cancer cells. 27. The method of any one of claims 1-26, wherein the immortalized cells are plated on an ultra-low attachment surface prior to the co-culturing step. 28. The method of any one of claims 1 to 27, wherein prior to the co-culturing step, the immortalized cells are plated in a multiwell plate, optionally in a 6-well, 24-well or 96-well plate. 29. The method of claim 27 or 28, wherein the immortalized cells are plated at a density of about 5,000 to about 10,000 live cells/culture. 30. The method of any one of claims 27-29, wherein the ultra-low adhesion surface comprises a hydrophilic neutrally charged hydrogel coating. 31. The method of any one of claims 27-30, wherein the immortalized cells are cultured for preferably about 2 to 6 days, more preferably for about 4 days, to produce three-dimensional spheroids. 32. The method of any one of claims 1-31, wherein prior to the co-culturing step, the TILs are added to the culture of the immortalized cells at an E:T ratio of about 20:1 to 1:1. 33. The method of any one of claims 1 to 32, wherein the co-culturing step lasts about 4 hours, about 8 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 3 days, about 4 hours. days or about 5 days, wherein optionally the co-culturing step is for about 1 day to about 5 days. 34. The method according to any one of claims 1 to 33, wherein the step of assessing efficacy comprises the release of cytokines from the immortalized cells, optionally the release of IFN-γ, IL-2, TNFα and/or IL-6, A method comprising measuring cell death and/or viability of an immortalized cell. 35. The method of any one of claims 1-34, wherein assessing efficacy comprises measuring growth, cell death and/or viability of the immortalized cells. The method of any one of claims 1 to 35, wherein the measuring step includes real-time cell viability assay, ATP cell viability assay, live cell protease viability assay, tetrazolium reduced cell viability. Performing an assay selected from the following assays, resazurin reduced cell viability assay, dead-cell protease release cytotoxicity assay, lactate dehydrogenase release cytotoxicity assay, and DNA dye cytotoxicity assay. Including, method. A method of assessing the potency of tumor infiltrating lymphocytes (TIL), comprising:
co-culturing a clonal population of TILs and engineered cancer cells, wherein the engineered cancer cells express an anti-CD3 antibody or an anti-CD3 antibody fragment; and
Assessing death and/or viability of the engineered cancer cells
Method, including. 38. The method of claim 37, wherein the TIL expresses CD3 antigen. 39. The method of claim 37 or 38, wherein the engineered cancer cells express an anti-CD3 antibody fragment. 40. The method of claim 39, wherein the anti-CD3 antibody fragment is an anti-CD3 single chain variable fragment (scFv). 41. The method of claim 40, wherein the anti-CD3 scFv is mOKT3 scFv. 42. The method of claim 41, wherein the mOKT3 scFv is a low affinity mOKT3 scFv variant. 43. The method of claim 42, wherein the low affinity mOKT3 scFv variant binds CD3 with a KD that is lower than the dissociation constant (KD) of the mOKT3 scFv, wherein the KD of the mOKT3 scFv is about 5×10 ^-10 M. 44. The method of claim 43, wherein the low affinity mOKT3 scFv variant binds CD3 with a KD that is at least 1000-fold lower than the dissociation constant KD of mOKT3 scFv. 45. The method of claim 43 or 44, wherein the low affinity mOKT3 scFv variant comprises an amino acid sequence with R55L and Y57T mutations compared to the amino acid sequence of SEQ ID NO:2. 46. The method of any one of claims 37 to 45, wherein the anti-CD3 antibody or anti-CD3 antibody fragment is a membrane-tethered anti-CD3 antibody or a membrane tethered anti-CD3 antibody fragment, respectively. 47. The method of any one of claims 37-46, wherein the TIL is an engineered TIL (eTIL). 48. The method of claim 47, wherein the eTIL is an edited eTIL. 49. The method of claim 48, wherein the edited eTIL comprises a genomic modification. 50. The method of any one of claims 37-49, wherein the engineered cancer cells are selected from engineered melanoma cells, engineered colorectal cancer cells, engineered cholangiocarcinoma cells, and engineered breast cancer cells. 51. The method of claim 50, wherein the engineered cancer cells are engineered melanoma cells. 52. The method of any one of claims 37-51, wherein said co-culturing is for at least 24 hours, optionally for about 24 to 72 hours. The method of any one of claims 37 to 52, wherein the measuring step includes real-time cell viability assay, ATP cell viability assay, live cell protease viability assay, tetrazolium reduced cell viability assay, resazurin reduced cell viability. A method comprising performing an assay selected from a sex assay, an apoptotic cell protease release cytotoxicity assay, a lactate dehydrogenase release cytotoxicity assay, and a DNA dye cytotoxicity assay. A method of assessing the potency of tumor infiltrating lymphocytes (TIL), comprising:
co-culturing TILs, immortalized cells, and a bispecific molecule that activates and binds to T cells; and
Steps to evaluate the effectiveness of the TIL
Method, including. 55. The method of claim 54, wherein the TIL expresses CD3 antigen. 56. The method of claim 54 or 55, wherein the bispecific molecule comprises a molecule that binds CD3. 57. The method of any one of claims 54-56, wherein the TIL is an engineered TIL (eTIL). 58. The method of claim 57, wherein the eTIL is an edited eTIL. 59. The method of claim 58, wherein the edited eTIL comprises a genomic modification. 60. The method of any one of claims 54-59, wherein the immortalized cells comprise a dendritic population of immortalized cells. 61. The method of any one of claims 54-60, wherein the immortalized cells are human cells. 62. The method of any one of claims 54-61, wherein the immortalized cells are cancer cells. 63. The method of claim 62, wherein the cancer cells are selected from melanoma cells, colorectal cancer cells, cholangiocarcinoma cells, and breast cancer cells. 64. The method of claim 62 or 63, wherein the bispecific molecule comprises a molecule that binds CD19. 65. The method of claim 64, wherein the bispecific molecule is CD19-CD3 BiTE®. 66. The method of any one of claims 54-65, wherein the co-culturing step is for at least 24 hours, optionally for about 24 to 72 hours. The method of any one of claims 54 to 66, wherein the measuring step includes real-time cell viability assay, ATP cell viability assay, live cell protease viability assay, tetrazolium reduced cell viability assay, resazurin reduced cell viability. A method comprising performing an assay selected from a sex assay, an apoptotic cell protease release cytotoxicity assay, a lactate dehydrogenase release cytotoxicity assay, and a DNA dye cytotoxicity assay. As a method for evaluating the potency of polyclonal T cells,
co-culturing a polyclonal T cell and an immortalized cell, wherein the immortalized cell comprises a molecule that activates a T cell; and
Evaluating the effectiveness of the polyclonal T cells
Method, including. 69. The method of claim 68, wherein the molecule binds a T cell antigen. 70. The method of claim 69, wherein the polyclonal T cells express the T cell antigen. 71. The method of claim 69 or 70, wherein the T cell antigen is a CD3 antigen. 72. The method of any one of claims 1-71, wherein the immortalized cell expresses the molecule. 73. The method of any one of claims 1-72, wherein the molecule is an antibody or antibody fragment. 74. The method of claim 73, wherein the antibody fragment is selected from single chain variable fragment (scFv), F(ab')2 fragment, Fab fragment, Fab' fragment and Fv fragment. 75. The method of claim 74, wherein the antibody fragment is an scFv. 76. The method of any one of claims 73-75, wherein the antibody or antibody fragment is an OKT3 antibody or OKT3 antibody fragment, respectively. 77. The method of claim 76, wherein the OKT3 antibody fragment is membrane bound OKT3 (mOKT3) scFv. 78. The method of claim 77, wherein the mOKT3 scFv is a low affinity mOKT3 scFv variant. 79. The method of any one of claims 71 to 78, wherein the molecule binds CD3 with a KD that is higher than the dissociation constant (KD) of the mOKT3 scFv, wherein the KD of the mOKT3 scFv is about 5×10 ^-10 M. method. The method of claim 78 or 79, wherein the low affinity mOKT3 scFv variant comprises an amino acid sequence with R55 and Y57 mutations compared to the amino acid sequence of SEQ ID NO:2. 81. The method of claim 80, wherein the low affinity mOKT3 scFv variant comprises an amino acid sequence with R55M and Y57A mutations compared to the amino acid sequence of SEQ ID NO:2. 82. The method of claim 81, wherein the low affinity mOKT3 scFv variant binds CD3 with a KD that is at least 250-fold higher than the dissociation constant KD of mOKT3 scFv. 75. The method of claim 74, wherein the low affinity mOKT3 scFv variant comprises an amino acid sequence with R55L and Y57T relative to the amino acid sequence of SEQ ID NO:2. 84. The method of claim 83, wherein the low affinity mOKT3 scFv variant binds CD3 with a KD that is at least 1000 times higher than the dissociation constant KD of the OKT3 scFv. 73. The method of any one of claims 68 to 72, wherein said molecule is selected from bacterial superantigens, optionally Staphylococcus enterotoxin B (SEB), phytohemagglutinin (PHA) and concanavalin A (ConA). Chosen method. 86. The method of any one of claims 1-85, wherein the molecule is a membrane-tethered molecule. 69. The method of claim 68, wherein the polyclonal T cells comprise neoantigen-specific T cells. 69. The method of claim 68, wherein the polyclonal T cells are derived from peripheral blood. 69. The method of claim 68, wherein the polyclonal T cells are derived from bone marrow. 89. The method of any one of claims 1-89, wherein the immortalized cells comprise a genetic population of immortalized cells. 91. The method of any one of claims 1-90, wherein the immortalized cells are immortalized human cells. 92. The method of any one of claims 1-91, wherein the immortalized cells are engineered cancer cells. 93. The method of any one of claims 1-92, wherein the engineered cancer cells are selected from engineered melanoma cells, engineered colorectal cancer cells, engineered cholangiocarcinoma cells, and engineered breast cancer cells. 94. The method of any one of claims 1 to 93, wherein the co-culturing step is for at least 4 hours. 95. The method of any one of claims 1 to 94, wherein assessing the efficacy comprises releasing cytokines from the immortalized cells, optionally releasing IFN-γ and/or IL-6, apoptosis of the immortalized cells, and /or a method comprising measuring viability. 96. The method of any one of claims 1-95, wherein assessing efficacy comprises measuring growth, cell death and/or viability of the immortalized cells. The method of any one of claims 1 to 96, wherein the measuring step includes real-time cell viability assay, ATP cell viability assay, live cell protease viability assay, tetrazolium reduced cell viability assay, resazurin reduced cell viability. A method comprising performing an assay selected from a sex assay, an apoptotic cell protease release cytotoxicity assay, a lactate dehydrogenase release cytotoxicity assay, and a DNA dye cytotoxicity assay. As a method for evaluating the potency of polyclonal T cells,
co-culturing polyclonal T cells, immortalized cells, and a bispecific molecule that activates the T cells and binds to the immortalized cells; and
Evaluating the effectiveness of the polyclonal T cells
Method, including. 99. The method of claim 98, wherein the polyclonal T cells express CD3 antigen. 100. The method of claim 98 or 99, wherein the bispecific molecule comprises a molecule that binds CD3. 99. The method of claim 98, wherein the polyclonal T cells comprise neoantigen-specific T cells. 100. The method of claim 99, wherein the polyclonal T cells are derived from peripheral blood. 100. The method of claim 99, wherein the polyclonal T cells are derived from bone marrow. 103. The method of any one of claims 98-102, wherein the immortalized cells comprise a degenerative population of immortalized cells. 105. The method of any one of claims 98-104, wherein the immortalized cells are human cells. 106. The method of any one of claims 98-105, wherein the immortalized cells are cancer cells. 107. The method of claim 106, wherein the cancer cells are selected from melanoma cells, colorectal cancer cells, cholangiocarcinoma cells, and breast cancer cells. 108. The method of claim 106 or 107, wherein the bispecific molecule comprises a molecule that binds CD19. 109. The method of claim 108, wherein the bispecific molecule is CD19-CD3 BiTE®. 110. The method of any one of claims 98-109, wherein said co-culturing is for at least 24 hours, optionally for about 24 to 72 hours. The method of any one of claims 98 to 110, wherein the measuring step comprises real-time cell viability assay, ATP cell viability assay, live cell protease viability assay, tetrazolium reduced cell viability assay, resazurin reduced cell viability. A method comprising performing an assay selected from a sex assay, an apoptotic cell protease release cytotoxicity assay, a lactate dehydrogenase release cytotoxicity assay, and a DNA dye cytotoxicity assay.